Compositions and methods for the treatment and diagnosis of cardiovascular disease using rchd534 as a target

ABSTRACT

The present invention relates to methods and compositions for the treatment and diagnosis of cardiovascular disease, including, but not limited to, atherosclerosis, ischemia/reperfusion, hypertension, restenosis, and arterial inflammation. Specifically, the present invention identifies and describes genes which are differentially expressed in cardiovascular disease states, relative to their expression in normal, or non-cardiovascular disease states, and/or in response to manipulations relevant to cardiovascular disease. Further, the present invention identifies and describes genes via the ability of their gene products to interact with gene products involved in cardiovascular disease. Still further, the present invention provides methods for the identification and therapeutic use of compounds as treatments of cardiovascular disease. Moreover, the present invention provides methods for the diagnostic monitoring of patients undergoing clinical evaluation for the treatment of cardiovascular disease, and for monitoring the efficacy of compounds in clinical trials. Additionally, the present invention describes methods for the diagnostic evaluation and prognosis of various cardiovascular diseases, and for the identification of subjects exhibiting a predisposition to such conditions.

[0001] This application is a continuation-in-part of co-pendingapplication Ser. No. 08/386,844, filed Feb. 10, 1995, which is herebyincorporated by reference in its entirety.

1. INTRODUCTION

[0002] The present invention relates to methods and compositions for thetreatment and diagnosis of cardiovascular disease, including, but notlimited to, atherosclerosis, ischemia/reperfusion, hypertension,restenosis, and arterial inflammation. Genes which are differentiallyexpressed in cardiovascular disease states, relative to their expressionin normal, or non-cardiovascular disease states are identified. Genesare also identified via the ability of their gene products to interactwith other gene products involved in cardiovascular disease. The genesidentified may be used diagnostically or as targets for therapeuticintervention. In this regard, the present invention provides methods forthe identification and therapeutic use of compounds in the treatment anddiagnosis of cardiovascular disease. Additionally, methods are providedfor the diagnostic monitoring of patients undergoing clinical evaluationfor the treatment of cardiovascular disease, for monitoring the efficacyof compounds in clinical trials, and for identifying subjects who may bepredisposed to cardiovascular disease.

2. BACKGROUND OF THE INVENTION

[0003] Cardiovascular disease is a major health risk throughout theindustrialized world. Atherosclerosis, the most prevalent ofcardiovascular diseases, is the principal cause of heart attack, stroke,and gangrene of the extremities, and thereby the principle cause ofdeath in the United States. Atherosclerosis is a complex diseaseinvolving many cell types and molecular factors (for a detailed review,see Ross, 1993, Nature 362: 801-809). The process, in normalcircumstances a protective response to insults to the endothelium andsmooth muscle cells (SMCs) of the wall of the artery, consists of theformation of fibrofatty and fibrous lesions or plaques, preceded andaccompanied by inflammation. The advanced lesions of atherosclerosis mayocclude the artery concerned, and result from an excessiveinflammatory-fibroproliferative response to numerous different forms ofinsult. For example, shear stresses are thought to be responsible forthe frequent occurrence of atherosclerotic plaques in regions of thecirculatory system where turbulent blood flow occurs, such as branchpoints and irregular structures.

[0004] The first observable event in the formation of an atheroscleroticplaque occurs when blood-borne monocytes adhere to the vascularendothelial layer and transmigrate through to the sub-endothelial space.Adjacent endothelial cells at the same time produce oxidized low densitylipoprotein (LDL). These oxidized LDL's are then taken up in largeamounts by the monocytes through scavenger receptors expressed on theirsurfaces. In contrast to the regulated pathway by which native LDL(nLDL) is taken up by nLDL specific receptors, the scavenger pathway ofuptake is not regulated by the monocytes.

[0005] These lipid-filled monocytes are called foam cells, and are themajor constituent of the fatty streak. Interactions between foam cellsand the endothelial and SMCs which surround them lead to a state ofchronic local inflammation which can eventually lead to smooth musclecell proliferation and migration, and the formation of a fibrous plaque.Such plaques occlude the blood vessel concerned and thus restrict theflow of blood, resulting in ischemia.

[0006] Ischemia is a condition characterized by a lack of oxygen supplyin tissues of organs due to inadequate perfusion. Such inadequateperfusion can have number of natural causes, including atheroscleroticor restenotic lesions, anemia, or stroke, to name a few. Many medicalinterventions, such as the interruption of the flow of blood duringbypass surgery, for example, also lead to ischemia. In addition tosometimes being caused by diseased cardiovascular tissue, ischemia maysometimes affect cardiovascular tissue, such as in ischemic heartdisease. Ischemia may occur in any organ, however, that is suffering alack of oxygen supply.

[0007] The most common cause of ischemia in the heart is atheroscleroticdisease of epicardial coronary arteries. By reducing the lumen of thesevessels, atherosclerosis causes an absolute decrease in myocardialperfusion in the basal state or limits appropriate increases inperfusion when the demand for flow is augmented. Coronary blood flow canalso be limited by arterial thrombi, spasm, and, rarely, coronaryemboli, as well as by ostial narrowing due to luetic aortitis.congenital abnormalities, such as anomalous origin of the left anteriordescending coronary artery from the pulmonary artery, may causemyocardial ischemia and infarction in infancy, but this cause is veryrare in adults. Myocardial ischemia can also occur if myocardial oxygendemands are abnormally increased, as in severe ventricular hypertrophydue to hypertension or aortic stenosis. The latter can be present withangina that is indistinguishable from that caused by coronaryatherosclerosis. A reduction in the oxygen-carrying capacity of theblood, as in extremely severe anemia or in the presence ofcarboxy-hemoglobin, is a rare cause of myocardial ischemia. Notinfrequently, two or more causes of ischemia will coexist, such as anincrease in oxygen demand due to left ventricular hypertrophy and areduction in oxygen supply secondary to coronary atherosclerosis.

[0008] The principal surgical approaches to the treatment of ischemicatherosclerosis are bypass grafting, endarterectomy, and percutaneoustranslumenal angioplasty (PCTA). The failure rate after these approachesdue to restenosis, in which the occlusions recur and often become evenworse, is extraordinarily high (30-50%). It appears that much of therestenosis is due to further inflammation, smooth muscle accumulation,and thrombosis.

[0009] Very recently, a modified balloon angioplasty approach was usedto treat arterial restenosis in pigs by gene therapy (Ohno et al., 1994,Science 265: 781-784). A specialized catheter was used to introduce arecombinant adenovirus carrying the gene encoding thymidine kinase (tk)into the cells at the site of arterial blockage. Subsequently, the pigswere treated with ganciclovir, a nucleoside analog which is converted bytk into a toxic form which kills cells when incorporated into DNA.Treated animals had a 50% to 90% reduction in arterial wall thickeningwithout any observed local or systemic toxicities. Because of thepresumed role of the excessive inflammatory-fibroproliferative responsein atherosclerosis and ischemia, a number of researchers haveinvestigated, in the context of arterial injury, the expression ofcertain factors involved in inflammation, cell recruitment andproliferation. These factors include growth factors, cytokines, andother chemicals, including lipids involved in cell recruitment andmigration, cell proliferation and the control of lipid and proteinsynthesis.

[0010] For example, the expression of PDGF (platelet derived growthfactor) or its receptor was studied: in rats during repair of arterialinjury (Majesky et al., 1990, J.

[0011] Cell Biol. 111: 2149); in adherent cultures of humanmonocyte-derived macrophages treated with oxidized LDL (Malden et al.,1991, J. Biol. Chem. 266: 13901); and in bovine aortic endothelial cellssubjected to fluid shear stress (Resnick et al., 1993, Proc. Natl. Acad.Sci. USA 90: 4591-4595). Expression of IGF-I (insulin-like growthfactor-I) was studied after balloon deendothelialization of rat aorta(Cercek et al., 1990, Circulation Research 66: 1755-1760).

[0012] Other studies have focused on the expression ofadhesion-molecules on the surface of activated endothelial cells whichmediate monocyte adhesion. These adhesion molecules includeintracellular adhesion molecule-1, ICAM-1 (Simmons et al., 1988, Nature,331: 624-627), ELAM (Bevilacqua et al., 1989, Science 243: 1160-1165;Bevilacqua et al., 1991, Cell 67: 233), and vascular cell adhesionmolecule, VCAM-1 (Osborn et al., 1989, Cell 59: 1203-1211); all of thesesurface molecules are induced transcriptionally in the presence of IL-1.Histological studies reveal that ICAM-1, ELAM and VCAM-1 are expressedon endothelial cells in areas of lesion formation in vivo (Cybulsky etal., 1991, Science 251: 788-791; 1991, Arterioscler. Thromb. 11: 1397a;Poston et al., 1992, Am. J. Pathol. 140: 665-673). VCAM-1 and ICAM-1were shown to be induced in cultured rabbit arterial endothelium, aswell as in cultured human iliac artery endothelial cells bylysophophatidylcholine, a major phospholipid component of atherogeniclipoproteins. (Kume et al., 1992, J. Clin. Invest. 90: 1138-1144).VCAM-1, ICAM-1, and class II major histocompatibility antigens werereported to be induced in response to injury to rabbit aorta (Tanaka, etal., 1993, Circulation 88: 1788-1803).

[0013] Recently, cytomegalovirus (CMV) has been implicated in restenosisas well as atherosclerosis in general (Speir, et al., 1994, Science 265:391-394). It was observed that the CMV protein IE84 apparentlypredisposes smooth muscle cells to increased growth at the site ofrestenosis by combining with and inactivating p53 protein, which isknown to suppress tumors in its active form.

[0014] The foregoing studies are aimed at defining the role ofparticular gene products presumed to be involved in the excessiveinflammatory-fibroproliferative response leading to atheroscleroticplaque formation. However, such approaches cannot identify the fullpanoply of gene products that are involved in the disease process, muchless identifying those which may serve as therapeutic targets for thediagnosis and treatment of various forms of cardiovascular disease.

3. SUMMARY OF THE INVENTION

[0015] The present invention relates to methods and compositions for thetreatment and diagnosis of cardiovascular disease, including but notlimited to, atherosclerosis, ischemia/reperfusion, hypertension,restenosis, and arterial inflammation. Specifically, genes areidentified and described which are differentially expressed incardiovascular disease states, relative to their expression in normal,or non-cardiovascular disease states. “Differential expression”, as usedherein, refers to both quantitative as well as qualitative differencesin the genes' temporal and/or tissue expression patterns. Differentiallyexpressed genes may represent “fingerprint genes,” and/or “targetgenes.” “Fingerprint gene,” as used herein, refers to a differentiallyexpressed gene whose expression pattern may be utilized as part of aprognostic or diagnostic cardiovascular disease evaluation, or which,alternatively, may be used in methods for identifying compounds usefulfor the treatment of cardiovascular disease. “Target gene”, as usedherein, refers to a differentially expressed gene involved incardiovascular disease such that modulation of the level of target geneexpression or of target gene product activity may act to ameliorate acardiovascular disease condition. Compounds that modulate target geneexpression or activity of the target gene product can be used in thetreatment of cardiovascular disease.

[0016] Further, “pathway genes” are defined via the ability of theirproducts to interact with other gene products involved in cardiovasculardisease. Pathway genes may also exhibit target gene and/or fingerprintgene characteristics. Although the genes described herein may bedifferentially expressed with respect to cardiovascular disease, and/ortheir products may interact with gene products important tocardiovascular disease, the genes may also be involved in mechanismsimportant to additional cardiovascular processes.

[0017] The invention includes the products of such fingerprint, target,and pathway genes, as well as antibodies to such gene products.Furthermore, the engineering and use of cell- and animal-based models ofcardiovascular disease to which such gene products may contribute arealso described.

[0018] The present invention encompasses methods for prognostic anddiagnostic evaluation of cardiovascular disease conditions, and for theidentification of subjects exhibiting a predisposition to suchconditions. Furthermore, the invention provides methods for evaluatingthe efficacy of drugs, and monitoring the progress of patients, involvedin clinical trials for the treatment of cardiovascular disease.

[0019] The invention also provides methods for the identification ofcompounds that modulate the expression of genes or the activity of geneproducts involved in cardiovascular disease, as well as methods for thetreatment of cardiovascular disease which may involve the administrationof such compounds to individuals exhibiting cardiovascular diseasesymptoms or tendencies. The invention is based, in part, on systematicsearch strategies involving in vivo and in vitro cardiovascular diseaseparadigms coupled with sensitive and high throughput gene expressionassays. In contrast to approaches that merely evaluate the expression ofa given gene product presumed to play a role in a disease process, thesearch strategies and assays used herein permit the identification ofall genes, whether known or novel, that are expressed or repressed inthe disease condition, as well as the evaluation of their temporalregulation and function during disease progression. This comprehensiveapproach and evaluation permits the discovery of novel genes and geneproducts, as well as the identification of an array of genes and geneproducts (whether novel or known) involved in novel pathways that play amajor role in the disease pathology. Thus, the invention allows one todefine targets useful for diagnosis, monitoring, rational drug screeningand design, and/or other therapeutic intervention.

[0020] In the working examples described herein, eight novel human genesare identified that are demonstrated to be differentially expressed indifferent cardiovascular disease states Additionally, the differentialexpression of three previously identified human genes is described. Theidentification of these genes and the characterization of theirexpression in particular disease states provide newly identified rolesin cardiovascular disease for both the novel genes and the known genes.

[0021] Bcl-2 and glutathione peroxidase are the products of known genesthat are shown herein to be down regulated in monocytes of patientsexposed to an atherogenic high fat/high cholesterol diet. Furthermore,counteracting the down-regulation of bcl-2 under atherogenic conditions,as described herein, may ameliorate atherosclerosis. Accordingly,methods are provided for the diagnosis, monitoring in clinical trials,and treatment of cardiovascular disease based upon the discoveriesherein regarding the expression patterns of bcl-2 and glutathioneperoxidase. Because these two genes were known to be involved inpreventing apoptosis, the discovery of their down-regulation underatherogenic conditions provides a novel, positive correlation betweenapoptosis and atherogenesis. Accordingly, methods provided herein fordiagnosing, monitoring, and treating cardiovascular disease may also bebased on a number of genes involved in the apoptotic pathway, includingbut not limited to ICE (IL-1 converting enzyme); Bad; BAG-1 (Bcl-2associated athanogene 1, Takayama et al., 1995, Cell 80: 279-284); BAX(Bcl-2associated X protein, Oltvai et al., 1993, Cell 74: 609-619);BclXL (Boise, et al., 1993, Cell 74: 597-608); BAK (Bcl-2 antagonistkiller, Farrow et al., 1995. Nature 374: 631-733); and Bcl-Xs (Tsujmotoet al., 1984, Science 226: 1097-1099). The cardiovascular diseases thatmay be so diagnosed, monitored in clinical trials, and treated includebut are not limited to atherosclerosis, ischemia/reperfusion, andrestenosis.

[0022] rchd005, rchd024, rchd032, and rchd036 are newly identified genesthat are each up-regulated in endothelial cells treated with IL-1.Accordingly, methods are provided for the diagnosis, monitoring inclinical trials, and treatment of cardiovascular disease based upon thediscoveries herein regarding the expression patterns of rchd005,rchd024, rchd032, and rchd036.

[0023] Endoperoxide synthase is a known gene, and rchd502, rchd523,rchd528, and rchd534 are newly identified genes that are eachup-regulated in endothelial cells subjected to shear stress.Accordingly, methods are provided for the diagnosis, monitoring inclinical trials, screening for therapeutically effective compounds, andtreatment of cardiovascular disease based upon the discoveries hereinregarding the expression patterns of endoperoxide synthase, rchd502,rchd523, rchd528, and rchd534.

[0024] More specifically, because each of these genes is up-regulatedeither by IL-1 (rchd005, rchd024, rchd032, and rchd036) or by shearstress (endoperoxide synthase, rchd502, rchd523, rchd528, and rchd534),treatment methods can be designed to reduce or eliminate theirexpression, particularly in endothelial cells. Alternatively, treatmentmethods include inhibiting the activity of the protein products of thesegenes. In addition, detecting expression of these genes in excess ofnormal expression provides for the diagnosis of cardiovascular disease.Furthermore, in testing the efficacy of compounds during clinicaltrials, a decrease in the level of the expression of these genescorresponds to a return from a disease condition to a normal state, andthereby indicates a positive effect of the compound. The cardiovasculardiseases that may be so diagnosed, monitored in clinical trials, andtreated include but are not limited to atherosclerosis,ischemia/reperfusion, hypertension, restenosis, and arterialinflammation.

[0025] The rchd523 gene can be a particularly useful target fortreatment methods as well as diagnostic and clinical monitoring methods.As a transmembrane protein, the rchd523 gene product is accessible fromthe cell surface. Accordingly, natural ligands, derivatives of naturalligands, and antibodies that bind to the rchd523 gene product can beutilized to inhibit its activity, or alternatively, to target thespecific destruction of cells that are in the disease state.Furthermore, the extracellular domains of the rchd523 gene productprovide especially efficient screening systems for identifying compoundsthat bind to the rchd523 gene product. Compounds that bind the receptordomain of the rchd523 gene product, for example, can be identified bytheir ability to mobilize Ca²⁺ and thereby produce a fluorescent signal,as described in Section 5.5.1, below.

[0026] Such an assay system can also be used to screen and identifyantagonists of the interaction between the rchd523gene product andligands that bind to the rchd523 gene product. For example, thecompounds can compete with the endogenous (i.e., natural) ligand for therchd523 gene product. The resulting reduction in the amount ofligand-bound rchd523 gene transmembrane protein will modulate theactivity of disease state cells, such as endothelial cells. Solubleproteins or peptides, such as peptides comprising one or more of theextracellular domains, or portions and/or analogs thereof of the rchd523gene product, including, for example, soluble fusion proteins such asIg-tailed fusion proteins, can be particularly useful for this purpose.

[0027] Similarly, antibodies that are specific to one or more of theextracellular domain of the rchd523 product provide for the readydetection of this target gene product in diagnostic tests or in clinicaltest monitoring. Accordingly, endothelial cells can be treated, eitherin vivo or in vitro, with such a labeled antibody to determine thedisease state of endothelial cells. Because the rchd523 gene product isup-regulated in endothelial cells under shear stress, its detectionpositively corresponds with cardiovascular disease.

[0028] The examples presented in Sections 6-9, below, demonstrate theuse of the cardiovascular disease paradigms of the invention to identifycardiovascular disease target genes.

[0029] The example presented in Section 10, below, demonstrates the useof fingerprint genes in diagnostics and as surrogate markers for testingthe efficacy of candidate drugs in basic research and in clinicaltrials.

[0030] The example presented in Section 11, below, demonstrates the useof fingerprint genes, particularly rchd523, in the imaging of a diseasedcardiovascular tissue. The example presented in Section 12, below,demonstrates the use of target genes, particularly rchd523, in screeningfor ligands of target gene product redptor domains, as well asantagonists of the ligand-receptor interaction.

4. DESCRIPTION OF THE FIGURES

[0031]FIG. 1. In vivo cholesterol differential display. mRNA preparedfrom human monocytes isolated from the blood of patients on differentdiets. cDNA prepared from one patient on a high fat diet/high serumcholesterol (lanes 1,2) and low fat diet/low serum cholesterol (lanes3,4) was displayed using the forward primer T₁₁XG and the reverse primerOP014 (agcatggctc). The DNA corresponding to marked band (#14) wasexcised and amplified for sequence analysis.

[0032]FIG. 2. Band #14 Northern blot analysis. A random primer-labeledband #14 probe was hybridized with a Northern blot prepared from thesame patient's monocytes used in differential display. An 8 kb band wasseen in the low fat/low cholesterol conditions, and not in the highfat/high cholesterol conditions.

[0033]FIG. 3. Quantitative RT-PCR analysis of mouse bcl-2 mRNA levels inapoE-deficient mice. Monocyte RNA from apoE-deficient and control micewas compared using primers for mouse bcl-2(for-cacccctggcatcttctccttcc/rev-atcctcccccagttcaccccatcc) shown in theupper panel and mouse Actin(for-cctgatagatgggcactgtgt/rev-gaacacggcattgtcactaact) shown in thelower panel. A 1:3 dilution series of each input cDNA was done in pairswith the left band in each pair deriving from wild-type cDNA and theright band from apoE-deficient cDNA.

[0034]FIG. 4. RT-PCR quantification of human glutathione peroxidase(HUMGPXP1) cDNA from human clinical samples cDNA prepared from RNAderived from blood monocytes of the same patient under a high fat diet(serum cholesterol level=200; top panel) and a low fat diet (serumcholesterol level=170; bottom panel). Dilution series of amplificationproducts using GPX1.3 primers derived from HUMGPXP1 sequences 1121-1142(for-aagtcgcgcccgcccctgaaat) and 1260-1237(rev-gatccctggccaccgtccgtctga) is shown in the left portion of eachpanel. Dilution series of amplification products using human actinprimers (for-accctgaagtacccat/rev-tagaagcatttgcggtg) is shown in theright portion of each panel. The HUMGPXP1 band decreased in intensityunder a high fat diet (compare top left to bottom left), whereas theactin control band was equally intense under each diet (compare topright to bottom right).

[0035]FIG. 5. IL-1 activated HUVEC differential display. mRNA preparedfrom control HUVEC (lanes 9,10), 1 hr. of 10 units/ml IL-1 treatment(lanes 7,8), or 6 hr. treatment (lanes 11,12), was used in differentialdisplay reactions with the forward primer OPE7 (agatgcagcc) and reverseprimer T₁₁XA, which is an equimolar mix of oligonucleotides where X isG, C, or A. The DNA corresponding to marked band, rchd005, was excisedand amplified for Northern analysis and subcloning.

[0036]FIG. 6. Northern blot analysis of endothelial IL-1induciblerchd005. 2 μg of total RNA from control, 1 hr. and 6 hr. samples waseluted on an agarose gel, blotted, and incubated with a ³²P labeledprobe prepared from amplified rchd005 sequences. The indicated bandmigrated with markers corresponding to approximately 7.5 kb.

[0037]FIG. 7. A Northern blot prepared from shear stressed RNA andhybridized with the same rchd005 probe detects a 7.5 kb bandup-regulated most strongly at 1 hr.

[0038]FIG. 8. Band rchd005 DNA sequence. The sequence was determined bysequencing the insert of pRCHD005, resulting from the ligation ofamplified rchd005 sequences into the TA cloning vector.

[0039]FIG. 9. IL-1 activated HUVEC differential display. mRNA preparedfrom control HUVEC (lanes 3,4), 1 hr. of 10 units/ml IL-1 treatment(lanes 1,2), or 6 hr. treatment (lanes 5,6), was used in differentialdisplay reactions with the forward primer OPG20 (tctccctcag) and reverseprimer T₁₁XC, which is an equimolar mix of oligonucleotides where X isG, C, or A. The DNA corresponding to marked band, rchd024, was excisedand amplified for Northern analysis and subcloning.

[0040]FIG. 10. Northern blot analysis of endothelial IL-1inducible bandrchd024. 2 μg of total RNA from control, 1 hr. and 6 hr. samples waseluted on an agarose gel, blotted, and incubated with a ³²P labeledprobe prepared from amplified band rchd024 sequences. The indicated bandmigrated with markers corresponding to approximately 10 kb.

[0041]FIG. 11. Shear stress Northern blot analysis of endothelial IL-1inducible band rchd024. A Northern blot prepared from shear stressed RNAand hybridized with the same rchd024 probe detected a 10 kb bandup-regulated most strongly at 6 hr.

[0042]FIG. 12. Band rchd024 DNA sequence. The sequence was determined bysequencing the insert of pRCHD024, resulting from the ligation ofamplified rchd024 sequences into the TA cloning vector.

[0043]FIG. 13. IL-1 activated HUVEC differential display for rchd032.mRNA prepared from control HUVEC (lanes 3,4), 1 hr. of 10 units/ml IL-1treatment (lanes 1,2), or 6 hr. treatment (lanes 5,6), was used indifferential display reactions with the forward primer OPI9 (tggagagcag)and reverse primer T₁₁XA, which is an equimolar mix of oligonucleotideswhere X is G, C, or A. The DNA corresponding to marked band, rchd032,was excised and amplified for Northern analysis and subcloning.

[0044]FIG. 14. RT-PCR quantification of rchd032 cDNA from IL-1 activatedHUVEC's cDNA prepared from RNA derived from control, 1 hr., and 6 hr.IL-1 activated HUVEC's. Shown in lanes 1,2, and 3 are a 5 fold dilutionseries of input cDNA amplified in the upper panel with rchd032 primers(for-atttataaaggggtaattcatta/rev-ttaaagccaatttcaaaataat), and in thelower panel with human actin primers(for-accctgaagtaccccat/rev-tagaagcatttgcggtg). A band at the 1:125dilution in lane 3 is visible in the 6 hr. sample but not in thecontrol.

[0045]FIG. 15. Band rchd032 DNA sequence. The sequence was determined bysequencing the insert of pRCHD0321 resulting from the ligation ofamplified rchdO32 sequences into the TA cloning vector.

[0046]FIG. 16. IL-1 activated HUVEC differential display for rchd036.mRNA prepared from control HUVEC (lanes 3,4), 1 hr. of 10 units/ml IL-1treatment (lanes 1,2), or 6 hr. treatment (lanes 5,6), was used indifferential display reactions with the forward primer OPI17(ggtggtgatg) and reverse primer T₁₁XC, which is an equimolar mix ofoligonucleotides where X is G, C, or A. The DNA corresponding to markedband, rchd036, was excised and amplified for Northern analysis andsubcloning.

[0047]FIG. 17. Northern blot analysis of endothelial IL-1inducible bandrchd036. 2 μg of total RNA from control (lane 1), 1 hr. (lane 2), and 6hr. (lane 3) samples was eluted on an agarose gel, blotted, andincubated with a ³²P labeled probe prepared from amplified band rchd036sequences. The indicated band migrated with markers corresponding toapproximately 8 kb.

[0048]FIG. 18. Band rchd036 DNA sequence. The sequence was determined bysequencing the insert of pRCHD036, resulting from the ligation ofamplified rchd036 sequences into the TA cloning vector.

[0049]FIG. 19. Laminar shear stress HUVEC differential display. mRNAprepared from control HUVEC (lanes 3,4), 1 hr. (lanes 1,2) of 10 dyn/cm2laminar shear stress treatment or 6 hr. treatment (lanes 5,6), was usedin differential display reactions with the forward primer OPE7(agatgcagcc) and reverse primer T₁₁XA, which is an equimolar mix ofoligonucleotides where X is G, C, or A. The DNA corresponding to markedband, rchd502, was excised and amplified for Northern analysis andsubcloning.

[0050]FIG. 20. Northern blot analysis of shear stress inducible bandrchd502. 2 μg of total RNA from control, 1 hr. and 6 hr. shear stressedsamples was eluted on an agarose gel, blotted, and incubated with a ³²Plabeled probe prepared from amplified band rchd502 sequences. Theindicated band migrates with markers corresponding to approximately 4.5kb.

[0051]FIG. 21. Northern blot analysis of shear stress inducible bandrchd502 on IL-1 blot. 2 μg of total RNA from control (lane 1), 1 hr.(lane 2), and 6 hr. (lane 3) IL-1 induced HUVEC samples was eluted on anagarose gel, blotted, and incubated with a ³²P labeled probe preparedfrom amplified band rchd502 sequences. A 4.5 kb band is seen which wasnot up-regulated by IL-1.

[0052]FIG. 22. Band rchd502 DNA sequence. The sequence was determined bysequencing the insert of pRCHD502, resulting from the ligation ofamplified rchd502 sequences into the TA cloning vector.

[0053]FIG. 23. Laminar shear stress HUVEC differential display forrchd505. mRNA prepared from control HUVEC (lanes 3,4), 1 hr. (lanes 1,2)or 6 hr. (lanes 5,6) of 10 dyn/cm2 laminar shear stress treatment wasused in differential display reactions with the forward primer OPE2(ggtgcgggaa) and reverse primer T₁₁XA, which is an equimolar mix ofoligonucleotides where X is G,C, or A. The DNA corresponding to markedband, rchd505, was excised and amplified for Northern analysis andsubcloning.

[0054]FIG. 24. Northern blot analysis of shear stress inducible bandrchd505. 2 μg of total RNA from control, 1 hr. and 6 hr. shear stressedsamples was eluted on an agarose gel, blotted, and incubated with a ³²Plabeled probe prepared from amplified band rchd505 sequences. Theindicated band migrated with markers corresponding to approximately 5.0kb.

[0055]FIG. 25. Northern blot analysis of shear stress inducible bandrchd505 on IL-1 blot. 2 μg of total RNA from control (lane 1), 1 hr.(lane 2), and 6 hr. (lane 3) IL-1 induced HUVEC samples was eluted on anagarose gel, blotted, and incubated with a ³²P labeled probe preparedfrom amplified band rchd505 sequences. A 5.0 kb inducible band is seen.

[0056]FIG. 26. Laminar shear stress HUVEC differential display forrchd523. mRNA prepared from control HUVEC (lanes 3,4), 1 hr. (lanes 1,2)or 6 hr. (lanes 5,6) of 10 dyn/cm2 laminar shear stress treatment wasused in differential display reactions with the forward primer OPI11(acatgccgtg) and reverse primer T₁₁XC, which is an equimolar mix ofoligonucleotides where X is G,C, or A. The DNA corresponding to markedband, rchd523, was excised and amplified for Northern analysis andsubcloning.

[0057]FIG. 27. RT-PCR quantification of rchd523 cDNA from shear stressedendothelial cell cDNA prepared from RNA derived from control, 1 hr., and6 hr. shear stressed HUVEc's. Shown in lanes 1,2, and 3 are a 5-folddilution series of input cDNA amplified in the upper panel with rchd523primers (for-atgccgtgtgggttagtc/rev-attttatgggaaggtttttaca), and inlanes 4 and 5, a 5-fold dilution series using human actin primers(for-accctgaagtaccccat/rev-tagaagcatttgcggtg). A band at the 1:5dilution in lane 2 is visible in the 6 hr. sample but not in thecontrol.

[0058]FIG. 28. DNA and encoded amino acid sequence of the rchd523 gene.

[0059]FIG. 29. Laminar shear stress HUVEC differential display forrchd528. mRNA prepared from control HUVEC (lanes 3,4), 1 hr. (lanes 1,2)or 6 hr. (lanes 5,6) of 10 dyn/cm2 laminar shear stress treatment wasused in differential display reactions with the forward primer OPI19(aatgcgggag) and reverse primer T₁₁XG, which is an equimolar mix ofoligonucleotides where X is G,C, or A. The DNA corresponding to markedband, rchd528, was excised and amplified for Northern analysis andsubcloning.

[0060]FIG. 30. Northern blot analysis of shear stress inducible bandrchd528. 2 μg of total RNA from control (lane 1), 1 hr. (lane 2), and 6hr. (lane 3) shear stressed samples was eluted on an agarose gel,blotted, and incubated with a ³²P labeled probe prepared from amplifiedband rchd528 sequences. The indicated band migrated with markerscorresponding to approximately 5.0 kb.

[0061]FIG. 31. Band rchd528 DNA sequence. The sequence was determined bysequencing the insert of pRCHD528, resulting from the ligation ofamplified rchd528 sequences into the TA cloning vector.

[0062]FIG. 32. Restriction map of plasmid pScR-bcl2. FIG. 33. Northernblot analysis of expression of rchd036 mRNA under shear stress. RNA wasprepared from HUVEC's that were untreated (control) and treated withshear stress for 1 hr. and 6 hr. The blot was probed with labeledrchdO36 DNA.

[0063]FIG. 34. Northern blot analysis of expression of rchd534 mRNAunder shear stress. RNA was prepared from HUVEC's that were untreated(control) and treated with shear stress for 1 hr. and 6 hr. The blot wasprobed with labeled rchd534 DNA.

[0064]FIG. 35. DNA and encoded amino acid sequence of the rchd534 gene.

5. DETAILED DESCRIPTION OF THE INVENTION

[0065] Methods and compositions for the diagnosis and treatment ofcardiovascular disease, including but not limited to atherosclerosis,ischemia/reperfusion, hypertension, restenosis, and arterialinflammation, are described. The invention is based, in part, on theevaluation of the expression and role of all genes that aredifferentially expressed in paradigms that are physiologically relevantto the disease condition. This permits the definition of diseasepathways and the identification of targets in the pathway that areuseful both diagnostically and therapeutically.

[0066] Genes, termed “target genes” and/or “fingerprint genes” which aredifferentially expressed in cardiovascular disease conditions, relativeto their expression in normal, or non-cardiovascular disease conditions,are described in Section 5.4. Additionally, genes, termed “pathwaygenes” whose gene products exhibit an ability to interact with geneproducts involved in cardiovascular disease are also described inSection 5.4. Pathway genes may additionally have fingerprint and/ortarget gene characteristics. Methods for the identification of suchfingerprint, target, and pathway genes are described in Sections 5.1,5.2, and 5.3.

[0067] Further, the gene products of such fingerprint, target, andpathway genes are described in Section 5.4.2, antibodies to such geneproducts are described in Section 5.4.3, as are cell- and animal-basedmodels of cardiovascular disease to which such gene products maycontribute, in Section 5.4.4.

[0068] Methods for the identification of compounds which modulate theexpression of genes or the activity of gene products involved incardiovascular disease are described in Section 5.5. Methods formonitoring the efficacy of compounds during clinical trials aredescribed in Section 5.5.4. Additionally described below, in Section5.6, are methods for the treatment of cardiovascular disease.

[0069] Also discussed below, in Section 5.8, are methods for prognosticand diagnostic evaluation of cardiovascular disease, including theidentification of subjects exhibiting a predisposition to this disease,and the imaging of cardiovascular disease conditions.

[0070] 5.1. Identification of Differentially Expressed Genes

[0071] This section describes methods for the identification of geneswhich are involved in cardiovascular disease, including but not limitedto atherosclerosis, ischemia/reperfusion, hypertension, restenosis, andarterial inflammation. Such genes may represent genes which aredifferentially expressed in cardiovascular disease conditions relativeto their expression in normal, or non-cardiovascular disease conditions.Such differentially expressed genes may represent “target” and/or“fingerprint” genes. Methods for the identification of suchdifferentially expressed genes are described, below, in this section.Methods for the further characterization of such differentiallyexpressed genes, and for their identification as target and/orfingerprint genes, are presented, below, in Section 5.3.

[0072] “Differential expression” as used herein refers to bothquantitative as well as qualitative differences in the genes' temporaland/or tissue expression patterns. Thus, a differentially expressed genemay have its expression activated or completely inactivated in normalversus cardiovascular disease conditions (e.g., treated with oxidizedLDL versus untreated), or under control versus experimental conditions.Such a qualitatively regulated gene will exhibit an expression patternwithin a given tissue or cell type which is detectable in either controlor cardiovascular disease subjects, but is not detectable in both.Alternatively, such a qualitatively regulated gene will exhibit anexpression pattern within a given tissue or cell type which isdetectable in either control or experimental subjects, but is notdetectable in both. “Detectable”, as used herein, refers to an RNAexpression pattern which is detectable via the standard techniques ofdifferential display, reverse transcriptase- (RT-) PCR and/or Northernanalyses, which are well known to those of skill in the art.

[0073] Alternatively, a differentially expressed gene may have itsexpression modulated, i.e., quantitatively increased or decreased, innormal versus cardiovascular disease states, or under control versusexperimental conditions. The degree to which expression differs innormal versus cardiovascular disease or control versus experimentalstates need only be large enough to be visualized via standardcharacterization techniques, such as, for example, the differentialdisplay technique described below. Other such standard characterizationtechniques by which expression differences may be visualized include butare not limited to quantitative RT-PCR and Northern analyses.

[0074] Differentially expressed genes may be further described as targetgenes and/or fingerprint genes. “Fingerprint gene,” as used herein,refers to a differentially expressed gene whose expression pattern maybe utilized as part of a prognostic or diagnostic cardiovascular diseaseevaluation, or which, alternatively, may be used in methods foridentifying compounds useful for the treatment of cardiovasculardisease. A fingerprint gene may also have the characteristics of atarget gene.

[0075] “Target gene”, as used herein, refers to a differentiallyexpressed gene involved in cardiovascular disease in a manner by whichmodulation of the level of target gene expression or of target geneproduct activity may act to ameliorate symptoms of cardiovasculardisease. A target gene may also have the characteristics of afingerprint gene.

[0076] A variety of methods may be utilized for the identification ofgenes which are involved in cardiovascular disease. These methodsinclude but are not limited to the experimental paradigms described,below, in Section 5.1.1. Material from the paradigms may becharacterized for the presence of differentially expressed genesequences as discussed, below, in Section 5.1.2.

[0077] 5.1.1. Paradigms for the Identification of DifferentiallyExpressed Genes

[0078] One strategy for identifying genes that are involved incardiovascular disease is to detect genes that are expresseddifferentially under conditions associated with the disease versusnon-disease conditions. The sub-sections below describe a number ofexperimental systems, called aradigms, which may be used to detect suchdifferentially expressed genes. In general, the paradigms include atleast one experimental condition in which subjects or samples aretreated in a manner associated with cardiovascular disease, in additionto at least one experimental control condition lacking such diseaseassociated treatment. Differentially expressed genes are detected, asdescribed herein, below, by comparing the pattern of gene expressionbetween the experimental and control conditions.

[0079] Once a particular gene has been identified through the use of onesuch paradigm, its expression pattern may be further characterized bystudying its expression in a different paradigm. A gene may, forexample, be regulated one way in a given paradigm (e.g., up-regulation),but may be regulated differently in some other paradigm (e.g.,down-regulation). Furthermore, while different genes may have similarexpression patterns in one paradigm, their respective expressionpatterns may differ from one another under a different paradigm. Suchuse of multiple paradigms may be useful in distinguishing the roles andrelative importance of particular genes in cardiovascular disease.

[0080] 5.1.1.1. Foam Cell Paradigm—1

[0081] Among the paradigms which may be utilized for the identificationof differentially expressed genes involved in atherosclerosis, forexample, are paradigms designed to analyze those genes which may beinvolved in foam cell formation. Such paradigms may serve to identifygenes involved in the differentiation of this cell type, or their uptakeof oxidized LDL.

[0082] One embodiment of such a paradigm, hereinafter referred to asParadigm A. First, human blood is drawn and peripheral monocytes areisolated by methods routinely practiced in the art. These humanmonocytes can then be used immediately or cultured in vitro, usingmethods routinely practiced in the art, for 5 to 9 days where theydevelop more macrophage-like characteristics such as the up-regulationof scavenger receptors. These cells are then treated for various lengthsof time with agents thought to be involved in foam cell formation. Theseagents include but are not limited to oxidized LDL, acetylated LDL,lysophosphatidylcholine, and homocysteine. Control monocytes that areuntreated or treated with native LDL are grown in parallel. At a certaintime after addition of the test agents, the cells are harvested andanalyzed for differential expression as described in detail in Section5.1.2., below. The Example presented in Section 6, below, demonstratesin detail the use of such a foam cell paradigm to identify genes whichare differentially expressed in treated versus control cells.

[0083] 5.1.1.2. Foam Cell Paradigm—2

[0084] Alternative paradigms involving monocytes for detectingdifferentially expressed genes associated with atherosclerosis involvethe simulation of the phenomenon of transmigration. When monocytesencounter arterial injury, they adhere to the vascular endotheliallayer, transmigrate across this layer, and locate between theendothelium and the layer of smooth muscle cells that ring the artery.This phenomenon can be mimicked in vitro by culturing a layer ofendothelial cells isolated, for example, from human umbilical cord. Oncethe endothelial monolayer forms, monocytes drawn from peripheral bloodare cultured on top of the endothelium in the presence and absence ofLDL. After several hours, the monocytes transmigrate through theendothelium and develop into foam-cells after 3 to 5 days when exposedto LDL. In this system, as in vivo, the endothelial cells carry out theoxidation of LDL which is then taken up by the monocytes. As describedin sub-section 5.1.2. below, the pattern of gene expression can then becompared between these foam cells and untreated monocytes.

[0085] 5.1.1.3. Foam Cell Paradigm—3

[0086] Yet another system includes the third cell type, smooth musclecell, that plays a critical role in atherogenesis (Navab et al., 1988,J. Clin. Invest., 82: 1853). In this system, a multilayer of humanaortic smooth uscle cells was grown on a micropore filter covered with agel layer of native collagen, and a monolayer of human aorticendothelial cells was grown on top of the collagen layer. Exposure ofthis coculture to human monocytes in the presence of chemotactic factorrFMLP resulted in monocyte attachment to the endothelial cells followedby migration across the endothelial monolayer into the collagen layer ofthe subendothelial space. This type of culture can also be treated withLDL to generate foam cells. The foam cells can then be harvested andtheir pattern of gene expression compared to that of untreated cells asexplained below in sub-section 5.1.2.

[0087] 5.1.1.4. in vivo Monocyte Paradigm

[0088] An alternative embodiment of such paradigms for the study ofmonocytes, hereinafter referred to as Paradigm B, involves differentialtreatment of human subjects through the dietary control of lipidconsumption. Such human subjects are held on a low fat/low cholesteroldiet for three weeks, at which time blood is drawn, monocytes areisolated according to the methods routinely practiced in the art, andRNA is purified, as described below, in sub-section 5.1.2. These samepatients are subsequently switched to a high fat/high cholesterol dietand monocyte RNA is purified again. The patients may also be fed athird, combination diet containing high fat/low cholesterol and monocyteRNA may be purified once again. The order in which patients receive thediets may be varied. The RNA derived from patients maintained on two ofthe diets, or on all three diets, may then be compared and analyzed fordifferential gene expression as, explained below in sub-section 5.1.2.The Example presented in Section 7, below, demonstrates the use of suchan in vivo monocyte paradigm to identify genes which are expresseddifferentially in monocytes of patients maintained on an atherogenicdiet versus their expression under a control diet. Such a paradigm mayalso be used in conjunction with an in vitro preliminary detectionsystem, as described in Section 7, below.

[0089] 5.1.1.5. Endothelial Cell—IL-1 Paradigm

[0090] In addition to the detection of differential gene expression inmonocytes, paradigms focusing on endothelial cells may be used to detectgenes involved in cardiovascular disease. In one such paradigm,hereinafter referred to as Paradigm C, human umbilical vein endothelialcells (HUVEC's) are grown in vitro. Experimental cultures are treatedwith human IL-1β, a factor known to be involved in the inflammatoryresponse, in order to mimic the physiologic conditions involved in theatherosclerotic state. Alternatively experimental HUVEC cultures may betreated with lysophosphatidylcholine, a major phospholipid component ofatherogenic lipoproteins or oxidized human LDL. Control cultures aregrown in the absence of these compounds.

[0091] After a certain period of exposure treatment, experimental andcontrol cells are harvested and analyzed for differential geneexpression as described in sub-section 5.1.2, below. The Examplepresented in Section 8, below, demonstrates the use of such an IL-1induced endothelial cell paradigm to identify sequences which aredifferentially expressed in treated versus control cells.

[0092] 5.1.1.6. Endothelial Cell—Shear Stress Paradigm

[0093] In another paradigm involving endothelial cells, hereinafterreferred to as Paradigm D, cultures are exposed to fluid shear stresswhich is thought to be responsible for the prevalence of atheroscleroticlesions in areas of unusual circulatory flow. Unusual blood flow alsoplays a role in the harmful effects of ischemia/reperfusion, wherein anorgan receiving inadequate blood supply is suddenly reperfused with anoverabundance of blood when the obstruction is overcome.

[0094] Cultured HUVEC monolayers are exposed to laminar sheer stress byrotating the culture in a specialized apparatus containing liquidculture medium (Nagel et al., 1994, J. Clin. Invest. 94: 885-891).Static cultures grown in the same medium serve as controls. After acertain period of exposure to shear stress, experimental and controlcells are harvested and analyzed for differential gene expression asdescribed in sub-section 5.1.2, below. The Example presented in Section9, below, demonstrates the use of such a shear stressed endothelial cellparadigm to identify sequences which are differentially expressed inexposed versus control cells.

[0095] In all such paradigms designed to identify genes which areinvolved in cardiovascular disease, including but not limited to thosedescribed above in Sections 5.1.1.1 through 5.1.1.6, compounds such asdrugs known to have an ameliorative effect on the disease symptoms maybe incorporated into the experimental system. Such compounds may includeknown therapeutics, as well as compounds that are not useful astherapeutics due to their harmful side effects. Test cells that arecultured as explained in the paradigms described in Sections 5.1.1.1through 5.1.1.6, for example, may be exposed to one of these compoundsand analyzed for differential gene expression with respect to untreatedcells, according to the methods described below in Section 5.1.2. Inprinciple, according to the particular paradigm, any cell type involvedin the disease may be treated at any stage of the disease process bythese compounds.

[0096] Test cells may also be compared to unrelated cells (e.g.,fibroblasts) that are also treated with the compound, in order to screenout generic effects on gene expression that might not be related to thedisease. Such generic effects might be manifest by changes in geneexpression that are common to the test cells and the unrelated cellsupon treatment with the compound. By these methods, the genes and geneproducts upon which these compounds act can be identified and used inthe assays described below to identify novel therapeutic compounds forthe treatment of cardiovascular disease.

[0097] 5.1.2. Analysis of Paradigm Material

[0098] In order to identify differentially expressed genes, RNA, eithertotal or mRNA, may be isolated from one or more tissues of the subjectsutilized in paradigms such as those described earlier in this Section.RNA samples are obtained from tissues of experimental subjects and fromcorresponding tissues of control subjects. Any RNA isolation techniquewhich does not select against the isolation of mRNA may be utilized forthe purification of such RNA samples. See, for example, Sambrook et al.,1989, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press,N.Y.; and Ausubel, F. M. et al., eds., 1987-1993, Current Protocols inMolecular Biology, John Wiley & Sons, Inc. New York, both of which areincorporated herein by reference in their entirety. Additionally, largenumbers of tissue samples may readily be processed using techniques wellknown to those of skill in the art, such as, for example, thesingle-step RNA isolation process of Chomczynski, P. (1989, U.S. Pat.No. 4,843,155), which is incorporated herein by reference in itsentirety.

[0099] Transcripts within the collected RNA samples which represent RNAproduced by differentially expressed genes may be identified byutilizing a variety of methods which are well known to those of skill inthe art. For example, differential screening (Tedder, T. F. et al.,1988, Proc. Natl. Acad. Sci. USA 85:208-212), subtractive hybridization(Hedrick, S. M. et al., 1984, Nature 308:149-153; Lee, S. W. et al.,1984, Proc. Natl. Acad. Sci. USA 88:2825), and, preferably, differentialdisplay (Liang, P., and Pardee, A. B., 1993, U.S. Pat. No. 5,262,311,which is incorporated herein by reference in its entirety), may beutilized to identify nucleic acid sequences derived from genes that aredifferentially expressed.

[0100] Differential screening involves the duplicate screening of a cDNAlibrary in which one copy of the library is screened with a total cellcDNA probe corresponding to the mRNA population of one cell type while aduplicate copy of the cDNA library is screened with a total cDNA probecorresponding to the mRNA population of a second cell type. For example,one cDNA probe may correspond to a total cell cDNA probe of a cell typederived from a control subject, while the second cDNA probe maycorrespond to a total cell cDNA probe of the same cell type derived froman experimental subject. Those clones which hybridize to one probe butnot to the other potentially represent clones derived from genesdifferentially expressed in the cell type of interest in control versusexperimental subjects.

[0101] Subtractive hybridization techniques generally involve theisolation of mRNA taken from two different sources, e.g., control andexperimental tissue, the hybridization of the mRNA or single-strandedcDNA reverse-transcribed from the isolated mRNA, and the removal of allhybridized, and therefore double-stranded, sequences. The remainingnon-hybridized, single-stranded cDNAs, potentially represent clonesderived from genes that are differentially expressed in the two mRNAsources. Such single-stranded cDNAs are then used as the startingmaterial for the construction of a library comprising clones derivedfrom differentially expressed genes. The differential display techniquedescribes a procedure, utilizing the well known polymerase chainreaction (PCR; the experimental embodiment set forth in Mullis, K. B.,1987, U.S. Pat. No. 4,683,202) which allows for the identification ofsequences derived from genes which are differentially expressed. First,isolated RNA is reverse-transcribed into single-stranded cDNA, utilizingstandard techniques which are well known to those of skill in the art.Primers for the reverse transcriptase reaction may include, but are notlimited to, oligo dT-containing primers, preferably of the reverseprimer type of oligonucleotide described below. Next, this techniqueuses pairs of PCR primers, as described below, which allow for theamplification of clones representing a random subset of the RNAtranscripts present within any given cell. Utilizing different pairs ofprimers allows each of the mRNA transcripts present in a cell to beamplified. Among such amplified transcripts may be identified thosewhich have been produced from differentially expressed genes.

[0102] The reverse oligonucleotide primer of the primer pairs maycontain an oligo dT stretch of nucleotides, preferably elevennucleotides long, at its 5′ end, which hybridizes to the poly(A) tail ofmRNA or to the complement of a cDNA reverse transcribed from an mRNApoly(A) tail. Second, in order to increase the specificity of thereverse primer, the primer may contain one or more, preferably two,additional nucleotides at its 3′ end. Because, statistically, only asubset of the mRNA derived sequences present in the sample of interestwill hybridize to such primers, the additional nucleotides allow theprimers to amplify only a subset of the mRNA derived sequences presentin the sample of interest. This is preferred in that it allows moreaccurate and complete visualization and characterization of each of thebands representing amplified sequences.

[0103] The forward primer may contain a nucleotide sequence expected,statistically, to have the ability to hybridize to cDNA sequencesderived from the tissues of interest. The nucleotide sequence may be anarbitrary one, and the length of the forward oligonucleotide primer mayrange from about 9 to about 13 nucleotides, with about 10 nucleotidesbeing preferred. Arbitrary primer sequences cause the lengths of theamplified partial cDNAs produced to be variable, thus allowing differentclones to be separated by using standard denaturing sequencing gelelectrophoresis.

[0104] PCR reaction conditions should be chosen which optimize amplifiedproduct yield and specificity, and, additionally, produce amplifiedproducts of lengths which may be resolved utilizing standard gelelectrophoresis techniques. Such reaction conditions are well known tothose of skill in the art, and important reaction parameters include,for example, length and nucleotide sequence of oligonucleotide primersas discussed above, and annealing and elongation step temperatures andreaction times.

[0105] The pattern of clones resulting from the reverse transcriptionand amplification of the mRNA of two different cell types is displayedvia sequencing gel electrophoresis and compared. Differences in the twobanding patterns indicate potentially differentially expressed genes.nce potentially differentially expressed gene sequences have beenidentified via bulk techniques such as, for example, those describedabove, the differential expression of such putatively differentiallyexpressed genes should be corroborated. Corroboration may beaccomplished via, for example, such well known techniques as Northernanalysis and/or RT-PCR.

[0106] Upon corroboration, the differentially expressed genes may befurther characterized, and may be identified as target and/orfingerprint genes, as discussed, below, in Section 5.3.

[0107] Also, amplified sequences of differentially expressed genesobtained through, for example, differential display may be used toisolate full length clones of the corresponding gene. The full lengthcoding portion of the gene may readily be isolated, without undueexperimentation, by molecular biological techniques well known in theart. For example, the isolated differentially expressed amplifiedfragment may be labeled and used to screen a cDNA library.Alternatively, the labeled fragment may be used to screen a genomiclibrary.

[0108] PCR technology may also be utilized to isolate full length cDNAsequences. As described, above, in this Section, the isolated, amplifiedgene fragments obtained through differential display have 5′ terminalends at some random point within the gene and have 3′ terminal ends at aposition preferably corresponding to the 3′ end of the transcribedportion of the gene. Once nucleotide sequence information from anamplified fragment is obtained, the remainder of the gene (i.e., the 5′end of the gene, when utilizing differential display) may be obtainedusing, for example, RT-PCR.

[0109] In one embodiment of such a procedure for the identification andcloning of full length gene sequences, RNA may be isolated, followingstandard procedures, from an appropriate tissue or cellular source. Areverse transcription reaction may then be performed on the RNA using anoligonucleotide primer complimentary to the mRNA that corresponds to theamplified fragment, for the priming of first strand synthesis. Becausethe primer is anti-parallel to the mRNA, extension will proceed towardthe 5′ end of the mRNA. The resulting RNA/DNA hybrid may then be“tailed” with guanines using a standard terminal transferase reaction,the hybrid may be digested with RNAase H, and second strand synthesismay then be primed with a poly-C primer. Using the two primers, the 5′portion of the gene is amplified using PCR. Sequences obtained may thenbe isolated and recombined with previously isolated sequences togenerate a full-length cDNA of the differentially expressed genes of theinvention. For a review of cloning strategies and recombinant DNAtechniques, see e.g., Sambrook et al., 1989, supra; and Ausubel et al.,1989, supra.

[0110] 5.2. Identification of Pathway Genes

[0111] This section describes methods for the identification of genes,termed “pathway genes”, involved in cardiovascular disease. “Pathwaygene”, as used herein, refers to a gene whose gene product exhibits theability to interact with gene products involved in cardiovasculardisease. A pathway gene may be differentially expressed and, therefore,may additionally have the characteristics of a target and/or fingerprintgene.

[0112] Any method suitable for detecting protein-protein interactionsmay be employed for identifying pathway gene products by identifyinginteractions between gene products and gene products known to beinvolved in cardiovascular disease. Such known gene products may becellular or extracellular proteins. Those gene products which interactwith such known gene products represent pathway gene products and thegenes which encode them represent pathway genes.

[0113] Among the traditional methods which may be employed areco-immunoprecipitation, crosslinking and co-purification throughgradients or chromatographic columns. Utilizing procedures such as theseallows for the identification of pathway gene products. Once identified,a pathway gene product may be used, in conjunction with standardtechniques, to identify its corresponding pathway gene. For example, atleast a portion of the amino acid sequence of the pathway gene productmay be ascertained using techniques well known to those of skill in theart, such as via the Edman degradation technique (see, e.g., Creighton,1983, Proteins: Structures and Molecular Principles, W. H. Freeman &Co., N.Y., pp.34-49). The amino acid sequence obtained may be used as aguide for the generation of oligonucleotide mixtures that can be used toscreen for pathway gene sequences. Screening made be accomplished, forexample by standard hybridization or PCR techniques. Techniques for thegeneration of oligonucleotide mixtures and screening are well-known.(See, e.g., Ausubel, supra., and PCR Protocols: A Guide to Methods andApplications, 1990, Innis, M. et al., eds. Academic Press, Inc., NewYork).

[0114] Additionally, methods may be employed which result in thesimultaneous identification of pathway genes which encode the proteininteracting with a protein involved in cardiovascular disease. Thesemethods include, for example, probing expression libraries with labeledprotein known or suggested to be involved in cardiovascular disease,using this protein in a manner similar to the well known technique ofantibody probing of λgt₁₁ libraries.

[0115] One such method which detects protein interactions in vivo, thetwo-hybrid system, is described in detail for illustration only and notby way of limitation. One version of this system has been described(Chien et al., 1991, Proc. Natl. Acad. Sci. USA, 88:9578-9582) and iscommercially available from Clontech (Palo Alto, Calif.).

[0116] Briefly, utilizing such a system, plasmids are constructed thatencode two hybrid proteins: one consists of the DNA-binding domain of atranscription activator protein fused to a known protein, and the otherconsists of the activator protein's activation domain fused to anunknown protein that is encoded by a cDNA which has been recombined intothis plasmid as part of a cDNA library. The plasmids are transformedinto a strain of the yeast Saccharomyces cerevisiae that contains areporter gene (e.g., lacZ) whose regulatory region contains theactivator's binding sites. Either hybrid protein alone cannot activatetranscription of the reporter gene, the DNA-binding domain hybridbecause it does not provide activation function and the activationdomain hybrid because it cannot localize to the activator's bindingsites. Interaction of the two proteins reconstitutes the functionalactivator protein and results in expression of the reporter gene, whichis detected by an assay for the reporter gene product.

[0117] The two-hybrid system or related methodology may be used toscreen activation domain libraries for proteins that interact with aknown “bait” gene protein. Total genomic or cDNA sequences may be fusedto the DNA encoding an activation domain. Such a library and a plasmidencoding a hybrid of the bait gene protein fused to the DNA-bindingdomain may be cotransformed into a yeast reporter strain, and theresulting transformants may be screened for those that express thereporter gene. These colonies may be purified and the library plasmidsresponsible for reporter gene expression may be isolated. DNA sequencingmay then be used to identify the proteins encoded by the libraryplasmids.

[0118] For example, and not by way of limitation, the bait gene may becloned into a vector such that it is translationally fused to the DNAencoding the DNA-binding domain of the GAL4 protein. Also by way ofexample, for the isolation of genes involved in cardiovascular disease,previously isolated genes known or suggested to play a part incardiovascular disease may be used as the bait genes. These include butare not limited to the genes for bFGF, IGF-I, VEGF, IL-1, M-CSF, TGFβ,TGFα, TNFA, HB-EGF, PDGF, IFN-γ, and GM-CSF, to name a few.

[0119] A cDNA library of the cell line from which proteins that interactwith bait gene are to be detected can be made using methods routinelypracticed in the art. According to the particular system describedherein, for example, the cDNA fragments may be inserted into a vectorsuch that they are translationally fused to the activation domain ofGAL4. This library may be co-transformed along with the bait gene-GAL4fusion plasmid into a yeast strain which contains a lacZ gene driven bya promoter which contains the GAL4 activation sequence. A cDNA encodedprotein, fused to the GAL4 activation domain, that interacts with baitgene will reconstitute an active GAL4 protein and thereby driveexpression of the lacZ gene. Colonies which express lacZ may be detectedby their blue color in the presence of X-gal. The cDNA may then bepurified from these strains, and used to produce and isolate the baitgene-interacting protein using techniques routinely practiced in theart.

[0120] Once a pathway gene has been identified and isolated, it may befurther characterized as, for example, discussed below, in Section 5.3.

[0121] 5.3. Characterization of Differentially Expressed and PathwayGenes

[0122] Differentially expressed genes, such as those identified via themethods discussed, above, in Section 5.1.1, pathway genes, such as thoseidentified via the methods discussed, above, in Section 5.2, as well asgenes identified by alternative means, may be further characterized byutilizing, for example, methods such as those discussed herein. Suchgenes will be referred to herein as “identified genes”.

[0123] Analyses such as those described herein will yield informationregarding the biological function of the identified genes. An assessmentof the biological function of the differentially expressed genes, inaddition, will allow for their designation as target and/or fingerprintgenes. Specifically, any of the differentially expressed genes whosefurther characterization indicates that a modulation of the gene'sexpression or a modulation of the gene product's activity may amelioratecardiovascular disease will be designated “target genes”, as defined,above, in Section 5.1. Such target genes and target gene products, alongwith those discussed below, will constitute the focus of the compounddiscovery strategies discussed, below, in Section 5.5.

[0124] Any of the differentially expressed genes whose furthercharacterization indicates that such modulations may not positivelyaffect cardiovascular disease, but whose expression pattern contributesto a gene expression “fingerprint pattern” correlative of, for example,a cardiovascular disease condition will be designated a “fingerprintgene”. “Fingerprint patterns” will be more fully discussed, below, inSection 5.8. It should be noted that each of the target genes may alsofunction as fingerprint genes, as may all or a subset of the pathwaygenes.

[0125] It should further be noted that the pathway genes may also becharacterized according to techniques such as those described herein.Those pathway genes which yield information indicating that they aredifferentially expressed and that modulation of the gene's expression ora modulation of the gene product's activity may amelioratecardiovascular disease will be also be designated “target genes”. Suchtarget genes and target gene products, along with those discussed above,will constitute the focus of the compound discovery strategiesdiscussed, below, in Section 5.5.

[0126] It should be additionally noted that the characterization of oneor more of the pathway genes may reveal a lack of differentialexpression, but evidence that modulation of the gene's activity orexpression may, nonetheless, ameliorate cardiovascular disease symptoms.In such cases, these genes and gene products would also be considered afocus of the compound discovery strategies of Section 5.5, below.

[0127] In instances wherein a pathway gene's characterization indicatesthat modulation of gene expression or gene product activity may notpositively affect cardiovascular disease, but whose expression isdifferentially expressed and which contributes to a gene expressionfingerprint pattern correlative of, for example, a cardiovasculardisease state, such pathway genes may additionally be designated asfingerprint genes.

[0128] Among the techniques whereby the identified genes may be furthercharacterized, the nucleotide sequence of the identified genes, whichmay be obtained by utilizing standard techniques well known to those ofskill in the art, may be used to further characterize such genes. Forexample, the sequence of the identified genes may reveal homologies toone or more known sequence motifs which may yield informationregarding-the biological function of the identified gene product.

[0129] Second, an analysis of the tissue distribution of the mRNAproduced by the identified genes may be conducted, utilizing standardtechniques well known to those of skill in the art. Such techniques mayinclude, for example, Northern analyses and RT-PCR. Such analysesprovide information as to whether the identified genes are expressed intissues expected to contribute to cardiovascular disease. Such analysesmay also provide quantitative information regarding steady state mRNAregulation, yielding data concerning which of the identified genesexhibits a high level of regulation in, preferably, tissues which may beexpected to contribute to cardiovascular disease.

[0130] Such analyses may also be performed on an isolated cellpopulation of a particular cell type derived from a given tissue.Additionally, standard in situ hybridization techniques may be utilizedto provide information regarding which cells within a given tissueexpress the identified gene. Such analyses may provide informationregarding the biological function of an identified gene relative tocardiovascular disease in instances wherein only a subset of the cellswithin the tissue is thought to be relevant to cardiovascular disease.

[0131] Third, the sequences of the identified genes may be used,utilizing standard techniques, to place the genes onto genetic maps,e.g., mouse (Copeland & Jenkins, 1991, Trends in Genetics 7: 113-118)and human genetic maps (Cohen, et al., 1993, Nature 366: 698-701). Suchmapping information may yield information regarding the genes'importance to human disease by, for example, identifying genes which mapnear genetic regions to which known genetic cardiovascular diseasetendencies map.

[0132] Fourth, the biological function of the identified genes may bemore directly assessed by utilizing relevant in vivo and in vitrosystems. In vivo systems may include, but are not limited to, animalsystems which naturally exhibit cardiovascular disease predisposition,or ones which have been engineered to exhibit such symptoms, includingbut not limited to the apoE-deficient atherosclerosis mouse model (Plumpet al., 1992, Cell 71: 343-353). Such systems are discussed in Section5.4.4.1, below.

[0133] The use of such an in vivo system is described in detail in theexample provided in Section 7, below, confirming the role of the targetgene bcl-2 (see Table 1, in Section 5.4.1, below). Briefly, bcl-2expression first was shown to be down-regulated in the apoE-deficientatherosclerosis mouse model. Then, a transgenic mouse was engineeredbearing the human bcl-2 gene under the control of a promoter which isinduced in monocyte foam cells under atherogenic conditions. To test theeffect of the induction of bcl-2 under such conditions, the transgenicmouse is crossed with the apoE-deficient mouse. apoE-deficient progenybearing the highly expressible bcl-2 gene are then examined for plaqueformation and development. Reduction in plaque formation and developmentin these progeny confirms the effectiveness of intervening incardiovascular disease through this target gene.

[0134] In vitro systems may include, but are not limited to, cell-basedsystems comprising cell types known or suspected of involvement incardiovascular disease. Such systems are discussed in detail, below, inSection 5.4.4.2.

[0135] In further characterizing the biological function of theidentified genes, the expression of these genes may be modulated withinthe in vivo and/or in vitro systems, i.e., either over- orunderexpressed, and the subsequent effect on the system then assayed.Alternatively, the activity of the product of the identified gene may bemodulated by either increasing or decreasing the level of activity inthe in vivo and/or in vitro system of interest, and its subsequenteffect then assayed.

[0136] The information obtained through such characterizations maysuggest relevant methods for the treatment of cardiovascular diseaseinvolving the gene of interest. For example, treatment may include amodulation of gene expression and/or gene product activity.Characterization procedures such as those described herein may indicatewhere such modulation should involve an increase or a decrease in theexpression or activity of the gene or gene product of interest. Suchmethods of treatment are discussed, below, in Section 5.5.4.

[0137] 5.4. Differentially Expressed and Pathway Genes

[0138] Identified genes, which include but are not limited todifferentially expressed genes such as those identified in Section5.1.1, above, and pathway genes, such as those identified in Section5.2, above, are described herein. Specifically, the nucleic acidsequences and gene products of such identified genes are describedherein. Further, antibodies directed against the identified genes'products, and cell- and animal-based models by which the identifiedgenes may be further characterized and utilized are also discussed inthis Section.

[0139] 5.4.1. Differentially Expressed and Pathway Gene Sequences

[0140] The differentially expressed and pathway genes of the inventionare listed below, in Table 1. Differentially expressed and pathway genenucleotide sequences are shown in FIGS. 8, 12, 15, 18, 22, 28, 31, and35.

[0141] Table 1 lists differentially expressed genes identified through,for example, the paradigms discussed, above, in Section 5.1.1, andbelow, in the examples presented in Sections 6 through 9. Table 1 alsosummarizes information regarding the further characterization of suchgenes.

[0142] First, the paradigm used initially to detect the differentiallyexpressed gene is described under the column headed “Paradigm ofOriginal Detection”. The expression patterns of those genes which havebeen shown to be differentially expressed, for example, under one ormore of the paradigm conditions described in Section 5.1.1 aresummarized under the column headed “Paradigm Expression Pattern”. Foreach of the tested genes, the paradigm which was used and the differencein the expression of the gene among the samples generated is shown. “

” indicates that gene expression is up-regulated (i.e., there is anincrease in the amount of detectable mRNA) among the samples generated,while “

” indicates that gene expression is down-regulated (i.e., there is adecrease in the amount of detectable mRNA) among the samples generated.“Detectable” as used herein, refers to levels of mRNA which aredetectable via, for example, standard Northern and/or RT-PCR techniqueswhich are well known to those of skill in the art.

[0143] Cell types in which differential expression was detected are alsosummarized in Table 1 under the column headed “Cell Type Detected in”.The column headed “Chromosomal Location” provides the human chromosomenumber on which the gene is located. Additionally, in instances whereinthe genes contain nucleotide sequences similar or homologous tosequences found in nucleic acid databases, references to suchsimilarities are listed.

[0144] The genes listed in Table 1 may be obtained using cloning methodswell known to those skilled in the art, including but not limited to theuse of appropriate probes to detect the genes within an appropriate cDNAor gDNA (genomic DNA) library. (See, for example, Sambrook et al., 1989,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratories,which is incorporated by reference herein in its entirety). Probes forthe novel sequences reported herein may be obtained directly from theisolated clones deposited with the NRRL, as indicated in Table 2, below.Alternatively, oligonucleotide probes for the novel genes may besynthesized based on the DNA sequences disclosed herein in FIGS. 8, 12,15, 18, 22, 28, 31, and 35. Such synthetic oligonucleotides may besimilarly produced based on the sequences provided for the previouslyknown genes described in the following references: Cleary et al., 1986,Cell 47: 19-28 (bcl-2); Takahashi et al., 1990, J. Biochem 108: 145-148(glutathione peroxidase); and Jones et al., 1993, J. Biol. Chem. 268:9049-9054 (prostaglandin endoperoxide synthase II), each of which isincorporated herein in its entirety.

[0145] The sequence obtained from clones containing partial codingsequences or non-coding sequences can be used to obtain the entirecoding region by using the RACE method (Chenchik, et al., 1995,CLONTECHniques (X) 1: 5-8; Barnes, 1994, Proc. Natl. Acad. Sci. USA 91:2216-2220; and Cheng et al., Proc. Natl. Acad. Sci. USA 91: 5695-5699).Oligonucleotides can be designed based on the sequence obtained from thepartial clone that can amplify a reverse transcribed mRNA encoding theentire coding sequence. This method was used, as described in theexample in Section 9, below, to obtain the entire coding region of therchd523 gene.

[0146] Alternatively, probes can be used to screen cDNA librariesprepared from an appropriate cell or cell line in which the gene istranscribed. For example, the genes described herein that were detectedin monocytes may be cloned from a cDNA library prepared from monocytesisolated as described in Section 7.1.1, below. In fact, as described indetail in the example in Section 9, below, this method was applied inorder to obtain the entire coding region of the rchd534 gene. Briefly,the up-regulation of this gene was detected, under Paradigm D, inHUVEC's subjected to shear stress. Then, amplified partial sequence ofthe rchd534 gene was subcloned. The insert was then isolated and used toprobe a cDNA library prepared from shear stress treated HUVEC's. A cDNAclone containing the entire rchd534 coding region was detected,isolated, and sequenced. The genes described herein that were detectedin endothelial cells may also be cloned from a cDNA library constructedfrom endothelial cells isolated as described in Progress in Hemostasisand Thrombosis, Vol. 3, P. Spaet, editor, Grune & Stratton Inc., NewYork, 1-28.

[0147] Alternatively, the genes may be retrieved from a human placentacDNA library (Clontech Laboratories, Palo Alto, CA), according toTakahashi et al., 1990, supra; a HUVEC cDNA library as described inJones et al. 1993, supra; or an acute lymphoblastic leukemia (SUP-B2)cDNA library as described in Cleary et al., 1986, supra, for example.Genomic DNA libraries can be prepared from any source. TABLE 1Differentially Expressed and Pathway Genes Paradigm Paradigm of Expr.Cell Type Chromosomal Gene Seq. ID # Original Detection Pattern Detectedin Location Ref Seq. Band 14: B ⇓ Monocytes 1 bcl-2 (Section 5.1.1.4)Glutathione B ⇓ Monocytes 2 peroxidase rchd005 1 C

Endothelial New 3 FIG. 8  (Section 5.1.1.5) rchd024 2 C

Endothelial 4 New rchd032 3 C

Endothelial New rchd036 4 C

Endothelial 15  New rchd502 5 D

Endothelial New 4 (Section 5.1.1.6) rchd505: D

Endothelial 5 Endoperoxide synthase rchd523 6 D

Endothelial 7 New rchd528 7 D

Endothelial New rchd534 36  D

Endothelial 15  New

[0148] Table 2, below, lists isolated clones that contain sequences ofthe novel genes listed in Table 1. Such clones were produced fromamplified sequences of the indicated differential display band whichwere subcloned into the TA cloning vector (Invitrogen, San Diego,Calif.), as described in Section 6.1, below. Also listed in Table 2,below, are the strains deposited with the NRRL which contain each suchclone. Such strains were produced by transforming E. coli strain INVαF′(Invitrogen) with the indicated plasmid, as described in Section 6.1,below. The names of the plasmids containing the entire coding region ofa novel gene bear the prefix pFCHD, and the names of the strainscarrying these plasmids bear the prefix FCHD. TABLE 2 Plasmid CloneStrain Deposited Contained within GENE with NPRL Deposited Strainrchd005 RCHD005 pRCHD005 rchd024 RCHD024 pRCHD024 rchd032 RCHD032pRCHD032 rchd036 RCHD036 pRCHD036 rchd502 RCHD502 pRCHD502 rchd523RCHD523 pFCHD523 RCDH523 pRCHD523 rchd528 RCHD528 pRCHD528 rchd534FCHD534 pFCHD534

[0149] As used herein, “differentially expressed gene” (i.e. target andfingerprint gene) or “pathway gene” refers to (a) a gene containing atleast one of the DNA sequences disclosed herein (as shown in FIGS. 8,12, 15, 18, 22, 28, 31, and 35), or contained in the clones listed inTable 2, as deposited with the NRRL; (b) any DNA sequence that encodesthe amino acid sequence encoded by the DNA sequences disclosed herein(as shown in FIGS. 8, 12, 15, 18, 22, 28, 31, and 35), contained in theclones, listed in Table 2, as deposited with the NRRL or containedwithin the coding region of the gene to which the DNA sequencesdisclosed herein (as shown in FIGS. 8, 12, 15, 18, 22, 28, 31, and 35)or contained in the clones listed in Table 2, as deposited with theNRRL, belong; (c) any DNA sequence that hybridizes to the complement ofthe coding sequences disclosed herein, contained in the clones listed inTable 2, as deposited with the NRRL, or contained within the codingregion of the gene to which the DNA sequences disclosed herein (as shownin FIGS. 8, 12, 15, 18, 22, 28, 31, and 35) or contained in the cloneslisted in Table 2, as deposited with the NRRL, belong, under highlystringent conditions, e.g., hybridization to filter-bound DNA in 0.5 MNaHPO_(4, 7)% sodium dodecyl sulfate (SDS), 1 mM EDTA at 65° C., andwashing in 0.1× SSC/0.1% SDS at 68° C. (Ausubel F. M. et al., eds.,1989, Current Protocols in Molecular Biology, Vol. I, Green PublishingAssociates, Inc., and John Wiley & sons, Inc., New York, at p. 2.10.3)and encodes a gene product functionally equivalent to a gene productencoded by sequences contained within the clones listed in Table 2;and/or (d) any DNA sequence that hybridizes to the complement of thecoding sequences disclosed herein, (as shown in FIGS. 8, 12, 15, 18, 22,28, 31, and 35) contained in the clones listed in Table 2, as depositedwith the NRRL or contained within the coding region of the gene to whichDNA sequences disclosed herein (as shown in FIGS. 8, 12, 15, 18, 22, 28,31, and 35) or contained in the clones, listed in Table 2, as depositedwith the NRRL, belong, under less stringent conditions, such asmoderately stringent conditions, e.g., washing in 0.2× SSC/0.1% SDS at42° C. (Ausubel et al., 1989, supra), yet which still encodes afunctionally equivalent gene product. The invention also includesnucleic acid molecules, preferably DNA molecules, that hybridize to, andare therefore the complements of, the DNA sequences (a) through (c), inthe preceding paragraph. Such hybridization conditions may be highlystringent or less highly stringent, as described above. In instanceswherein the nucleic acid molecules are deoxyoligonucleotides (“oligos”),highly stringent conditions may refer, e.g., to washing in 6× SSC/0.05%sodium pyrophosphate at 37° C. (for 14-base oligos), 48° C. (for 17-baseoligos), 55° C. (for 20-base oligos), and 60° C. (for 23-base oligos).These nucleic acid molecules may act as target gene antisense molecules,useful, for example, in target gene regulation and/or as antisenseprimers in amplification reactions of target gene nucleic acidsequences. Further, such sequences may be used as part of ribozymeand/or triple helix sequences, also useful for target gene regulation.Still further, such molecules may be used as components of diagnosticmethods whereby the presence of a cardiovascular disease-causing allele,may be detected.

[0150] The invention also encompasses (a) DNA vectors that contain anyof the foregoing coding sequences and/or their complements (i.e.,antisense); (b) DNA expression vectors that contain any of the foregoingcoding sequences operatively associated with a regulatory element thatdirects the expression of the coding sequences; and (c) geneticallyengineered host cells that contain any of the foregoing coding sequencesoperatively associated with a regulatory element that directs theexpression of the coding sequences in the host cell. As used herein,regulatory elements include but are not limited to inducible andnon-inducible promoters, enhancers, operators and other elements knownto those skilled in the art that drive and regulate expression. Theinvention includes fragments of any of the DNA sequences disclosedherein.

[0151] In addition to the gene sequences described above, homologues ofsuch sequences, as may, for example be present in other species, may beidentified and may be readily isolated, without undue experimentation,by molecular biological techniques well known in the art. Further, theremay exist genes at other genetic loci within the genome that encodeproteins which have extensive homology to one or more domains of suchgene products. These genes may also be identified via similartechniques. For example, the isolated differentially expressed genesequence may be labeled and used to screen a cDNA library constructedfrom mRNA obtained from the organism of interest. Hybridizationconditions will be of a lower stringency when the cDNA library wasderived from an organism different from the type of organism from whichthe labeled sequence was derived. Alternatively, the labeled fragmentmay be used to screen a genomic library derived from the organism ofinterest, again, using appropriately stringent conditions. Such lowstringency conditions will be well known to those of skill in the art,and will vary predictably depending on the specific organisms from whichthe library and the labeled sequences are derived. For guidanceregarding such conditions see, for example, Sambrook et al., 1989,Molecular Cloning, A Laboratory Manual, Cold Springs Harbor Press, N.Y.;and Ausubel et al., 1989, Current Protocols in Molecular Biology, GreenPublishing Associates and Wiley Interscience, N.Y.

[0152] Further, a previously unknown differentially expressed or pathwaygene-type sequence may be isolated by performing PCR using twodegenerate oligonucleotide primer pools designed on the basis of aminoacid sequences within the gene of interest. The template for thereaction may be cDNA obtained by reverse transcription of mRNA preparedfrom human or non-human cell lines or tissue known or suspected toexpress a differentially expressed or pathway gene allele.

[0153] The PCR product may be subcloned and sequenced to insure that theamplified sequences represent the sequences of a differentiallyexpressed or pathway gene-like nucleic acid sequence. The PCR fragmentmay then be used to isolate a full length cDNA clone by a variety ofmethods. For example, the amplified fragment may be labeled and used toscreen a bacteriophage cDNA library. Alternatively, the labeled fragmentmay be used to screen a genomic library.

[0154] PCR technology may also be utilized to isolate full length cDNAsequences. For example, RNA may be isolated, following standardprocedures, from an appropriate cellular or tissue source. A reversetranscription reaction may be performed on the RNA using anoligonucleotide primer specific for the most 5′ end of the amplifiedfragment for the priming of first strand synthesis. The resultingRNA/DNA hybrid may then be “tailed” with guanines using a standardterminal transferase reaction, the hybrid may be digested with RNAase H,and second strand synthesis may then be primed With a poly-C primer.Thus, cDNA sequences upstream of the amplified fragment may easily beisolated. For a review of cloning strategies which may be used, seee.g., Sambrook et al., 1989, supra.

[0155] In cases where the differentially expressed or pathway geneidentified is the normal, or wild type, gene, this gene may be used toisolate mutant alleles of the gene. Such an isolation is preferable inprocesses and disorders which are known or suspected to have a geneticbasis. Mutant alleles may be isolated from individuals either known orsuspected to have a genotype which contributes to cardiovascular diseasesymptoms. Mutant alleles and mutant allele products may then be utilizedin the therapeutic and diagnostic assay systems described below.

[0156] A cDNA of the mutant gene may be isolated, for example, by usingPCR, a technique which is well known to those of skill in the art. Inthis case, the first cDNA strand may be synthesized by hybridizing anoligo-dT oligonucleotide to mRNA isolated from tissue known or suspectedto be expressed in an individual putatively carrying the mutant allele,and by extending the new strand with reverse transcriptase. The secondstrand of the cDNA is then synthesized using an oligonucleotide thathybridizes specifically to the 5′ end of the normal gene. Using thesetwo primers, the product is then amplified via PCR, cloned into asuitable vector, and subjected to DNA sequence analysis through methodswell known to those of skill in the art. By comparing the DNA sequenceof the mutant gene to that of the normal gene, the mutation(s)responsible for the loss or alteration of function of the mutant geneproduct can be ascertained.

[0157] Alternatively, a genomic or cDNA library can be constructed andscreened using DNA or RNA, respectively, from a tissue known to orsuspected of expressing the gene of interest in an individual suspectedof or known to carry the mutant allele. The normal gene or any suitablefragment thereof may then be labeled and used as a probed td identifythe corresponding mutant allele in the library. The clone containingthis gene may then be purified through methods routinely practiced inthe art, and subjected to sequence analysis as described, above, in thisSection.

[0158] Additionally, an expression library can be constructed utilizingDNA isolated from or cDNA synthesized from a tissue known to orsuspected of expressing the gene of interest in an individual suspectedof or known to carry the mutant allele. In this manner, gene productsmade by the putatively mutant tissue may be expressed and screened usingstandard antibody screening techniques in conjunction with antibodiesraised against the normal gene product, as described, below, in Section5.4.3. (For screening techniques, see, for example, Harlow, E. and Lane,eds., 1988, “Antibodies: A Laboratory Manual”, Cold Spring Harbor Press,Cold Spring Harbor.) In cases where the mutation results in an expressedgene product with altered function (e.g., as a result of a missensemutation), a polyclonal set of antibodies are likely to cross-react withthe mutant gene product. Library clones detected via their reaction withsuch labeled antibodies can be purified and subjected to sequenceanalysis as described in this Section, above.

[0159] 5.4.2. Differentially Expressed and Pathway Gene Products

[0160] Differentially expressed and pathway gene products include thoseproteins encoded by the differentially expressed and pathway genesequences described in Section 5.4.1, above. Specifically,differentially expressed and pathway gene products may includedifferentially expressed and pathway gene polypeptides encoded by thedifferentially expressed and pathway gene sequences contained in theclones listed in Table 2, above, as deposited with the NRRL, orcontained in the coding regions of the genes to which DNA sequencesdisclosed herein (in FIGS. 8, 12, 15, 18, 22, 28, 31, and 35) orcontained in the clones, listed in Table 2, as deposited with the NRRL,belong, for example.

[0161] In addition, differentially expressed and pathway gene productsmay include proteins that represent functionally equivalent geneproducts. Such an equivalent differentially expressed or pathway geneproduct may contain deletions, additions or substitutions of amino acidresidues within the amino acid sequence encoded by the differentiallyexpressed or pathway gene sequences described, above, in Section 5.4.1,but which result in a silent change, thus producing a functionallyequivalent differentially expressed on pathway gene product. Amino acidsubstitutions may be made on the basis of similarity in polarity,charge, solubility, hydrophobicity, hydrophilicity, and/or theamphipathic nature of the residues involved.

[0162] For example, nonpolar (hydrophobic) amino acids include alanine,leucine, isoleucine, valine, proline, phenylalanine, tryptophan, andmethionine; polar neutral amino acids include glycine, serine,threonine, cysteine, tyrosine, asparagine, and glutamine; positivelycharged (basic) amino acids include arginine, lysine, and histidine; andnegatively charged (acidic) amino acids include aspartic acid andglutamic acid. “Functionally equivalent”, as utilized herein, refers toa protein capable of exhibiting a substantially similar in vivo activityas the endogenous differentially expressed or pathway gene productsencoded by the differentially expressed or pathway gene sequencesdescribed in Section 5.4.1, above. Alternatively, when utilized as partof assays such as those described, below, in Section 5.5, “functionallyequivalent” may refer to peptides capable of interacting with othercellular or extracellular molecules in a manner substantially similar tothe way in which the corresponding portion of the endogenousdifferentially expressed or pathway gene product would.

[0163] The differentially expressed or pathway gene products may beproduced by recombinant DNA technology using techniques well known inthe art. Thus, methods for preparing the differentially expressed orpathway gene polypeptides and peptides of the invention by expressingnucleic acid encoding differentially expressed or pathway gene sequencesare described herein. Methods which are well known to those skilled inthe art can be used to construct expression vectors containingdifferentially expressed or pathway gene protein coding sequences andappropriate transcriptional/translational control signals. These methodsinclude, for example, in vitro recombinant DNA techniques, synthetictechniques and in vivo recombination/genetic recombination. See, forexample, the techniques described in Sambrook et al., 1989, supra, andAusubel et al., 1989, supra. Alternatively, RNA capable of encodingdifferentially expressed or pathway gene protein sequences may bechemically synthesized using, for example, synthesizers. See, forexample, the techniques described in “Oligonucleotide Synthesis”, 1984,Gait, M. J. ed., IRL Press, Oxford, which is incorporated by referenceherein in its entirety. A variety of host-expression vector systems maybe utilized to express the differentially expressed or pathway genecoding sequences of the invention. Such host-expression systemsrepresent vehicles by which the coding sequences of interest may beproduced and subsequently purified, but also represent cells which may,when transformed or transfected with the appropriate nucleotide codingsequences, exhibit the differentially expressed or pathway gene proteinof the invention in situ. These include but are not limited tomicroorganisms such as bacteria (e.g., E. coli, B. subtilis) transformedwith recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expressionvectors containing differentially expressed or pathway gene proteincoding sequences; yeast (e.g. Saccharomyces, Pichia) transformed withrecombinant yeast expression vectors containing the differentiallyexpressed or pathway gene protein coding sequences; insect cell systemsinfected with recombinant virus expression vectors (e.g., baculovirus)containing the differentially expressed or pathway gene protein codingsequences; plant cell systems infected with recombinant virus expredionvectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus,TMV) or transformed with recombinant plasmid expression vectors (e.g.,Ti plasmid) containing differentially expressed or pathway gene proteincoding sequences; or mammalian cell systems (e.g. COS, CHO, BHK, 293,3T3) harboring recombinant expression constructs containing promotersderived from the genome of mammalian cells (e.g., metallothioneinpromoter) or from mammalian viruses (e.g., the adenovirus late promoter;the vaccinia virus 7.5K promoter).

[0164] In bacterial systems, a number of expression vectors may beadvantageously selected depending upon the use intended for thedifferentially expressed or pathway gene protein being expressed. Forexample, when a large quantity of such a protein is to be produced, forthe generation of antibodies or to screen peptide libraries, forexample, vectors which direct the expression of high levels of fusionprotein products that are readily purified may be desirable. Suchvectors include, but are not limited, to the E. coli expression vectorpUR278 (Ruther et al., 1983, EMBO J. 2:1791), in which thedifferentially expressed or pathway gene protein coding sequence may beligated individually into the vector in frame with the lac Z codingregion so that a fusion protein is produced; pIN vectors (Inouye &Inouye, 1985, Nucleic Acids Res. 13:3101-3109; Van Heeke & Schuster,1989, J. Biol. Chem. 264:5503-5509); and the like. pGEX vectors may alsobe used to express foreign polypeptides as fusion proteins withglutathione S-transferase (GST). In general, such fusion proteins aresoluble and can easily be purified from lysed cells by adsorption toglutathione-agarose beads followed by elution in the presence of freeglutathione. The pGEX vectors are designed to include thrombin or factorXa protease cleavage sites so that the cloned target gene protein can bereleased from the GST moiety.

[0165] In an insect system, Autographa califormica nuclear polyhedrosisvirus (AcNPV) is used as a vector to express foreign genes. The virusgrows in Spodoptera frugiperda cells. The differentially expressed orpathway gene coding sequence may be cloned individually intonon-essential regions (for example the polyhedrin gene) of the virus andplaced under control of an AcNPV promoter (for example the polyhedrinpromoter). Successful insertion of differentially expressed or pathwaygene coding sequence will result in inactivation of the polyhedrin geneand production of non-occluded recombinant virus (i.e., virus lackingthe proteinaceous coat coded for by the polyhedrin gene). Theserecombinant viruses are then used to infect Spodoptera frugiperda cellsin which the inserted gene is expressed. (E.g., see Smith et al., 1983,J. Virol. 46: 584; Smith, U.S. Pat. No. 4,215,051).

[0166] In mammalian host cells, a number of viral-based expressionsystems may be utilized. In cases where an adenovirus is used as anexpression vector, the differentially expressed or pathway gene codingsequence of interest may be ligated to an adenovirustranscription/translation control complex, e.g., the late promoter andtripartite leader sequence. This chimeric gene may then be inserted inthe adenovirus genome by in vitro or in vivo recombination. Insertion ina non-essential region of the viral genome (e.g., region E1 or E3) willresult in a recombinant virus that is viable and capable of expressingdifferentially expressed or pathway gene protein in infected hosts.(E.g., See Logan & Shenk, 1984, Proc. Natl. Acad. Sci. USA81:3655-3659). Specific initiation signals may also be required forefficient translation of inserted differentially expressed or pathwaygene coding sequences. These signals include the ATG initiation codonand adjacent sequences. In cases where an entire differentiallyexpressed or pathway gene, including its own initiation codon andadjacent sequences, is inserted into the appropriate expression vector,no additional translational control signals may be needed. However, incases where only a portion of the differentially expressed or pathwaygene coding sequence is inserted, exogenous translational controlsignals, including, perhaps, the ATG initiation codon, must be provided.Furthermore, the initiation codon must be in phase with the readingframe of the desired coding sequence to ensure translation of the entireinsert. These exogenous translational control signals and initiationcodons can be of a variety of origins, both natural and synthetic. Theefficiency of expression may be enhanced by the inclusion of appropriatetranscription enhancer elements, transcription terminators, etc. (seeBittner et al., 1987, Methods in Enzymol. 153:516-544).

[0167] In addition, a host cell strain may be chosen which modulates theexpression of the inserted sequences, or modifies and processes the geneproduct in the specific fashion desired. Such modifications (e.g.,glycosylation) and processing (e.g., cleavage) of protein products maybe important for the function of the protein. Different host cells havecharacteristic and specific mechanisms for the post-translationalprocessing and modification of proteins. Appropriate cell lines or hostsystems can be chosen to ensure the correct modification and processingof the foreign protein expressed. To this end, eukaryotic host cellswhich possess the cellular machinery for proper processing of theprimary transcript, glycosylation, and phosphorylation of the geneproduct may be used. Such mammalian host cells include but are notlimited to CHO, VERO, BHK, HeLa, COS, MDCK, 293, 3T3, WI38, etc.

[0168] For long-term, high-yield production of recombinant proteins,stable expression is preferred. For example, cell lines which stablyexpress the differentially expressed or pathway gene protein may beengineered. Rather than using expression vectors which contain viralorigins of replication, host cells can be transformed with DNAcontrolled by appropriate expression control elements (e.g., promoter,enhancer, sequences, transcription terminators, polyadenylation sites,etc.), and a selectable marker. Following the introduction of theforeign DNA, engineered cells may be allowed to grow for 1-2 days in anenriched media, and then are switched to a selective media. Theselectable marker in the recombinant plasmid confers resistance to theselection and allows cells to stably integrate the plasmid into theirchromosomes and grow to form foci which in turn can be cloned andexpanded into cell lines. This method may advantageously be used toengineer cell lines which express the differentially expressed orpathway gene protein. Such engineered cell lines may be particularlyuseful in screening and evaluation of compounds that affect theendogenous activity of the differentially expressed or pathway geneprotein.

[0169] A number of selection systems may be used, including but notlimited to the herpes simplex virus thymidine kinase (Wigler, et al.,1977, Cell 11:223), hypoxanthine-guanine phosphoribosyltransferase(Szybalska & Szybalski, 1962, Proc. Natl. Acad. Sci. USA 48:2026), andadenine phosphoribosyltransferase (Lowy, et al., 1980, Cell 22:817)genes can be employed in tk⁻, hgprt⁻ or aprt⁻ cells, respectively. Also,antimetabolite resistance can be used as the basis of selection fordhfr, which confers resistance to methotrexate (Wigler, et al., 1980,Natl. Acad. Sci. USA 77:3567; O'Hare, et al., 1981, Proc. Natl. Acad.Sci. USA 78:1527); gpt, which confers resistance to mycophenolic acid(Mulligan & Berg, 1981, Proc. Natl. Acad. Sci. USA 78:2072); neo, whichconfers resistance to the aminoglycoside G-418 (Colberre-Garapin, etal., 1981, J. Mol. Biol. 150:1); and hygro, which confers resistance tohygromycin (Santerre, et al., 1984, Gene 30:147) genes.

[0170] An alternative fusion protein system allows for the readypurification of non-denatured fusion proteins expressed in human celllines (Janknecht, et al., 1991, Proc. Natl. Acad. Sci. USA 88:8972-8976). In this system, the gene of interest is subcloned into avaccinia recombination plasmid such that the gene's open reading frameis translationally fused to an amino-terminal tag consisting of sixhistidine residues. Extracts from cells infected with recombinantvaccinia virus are loaded onto Ni²⁺ nitriloacetic acid-agarose columnsand histidine-tagged proteins are selectively eluted withimidazole-containing buffers.

[0171] When used as a component in assay systems such as thosedescribed, below, in Section 5.5, the differentially expressed orpathway gene protein may be labeled, either directly or indirectly, tofacilitate detection of a complex formed between the differentiallyexpressed or pathway gene protein and a test substance. Any of a varietyof suitable labeling systems may be used including but not limited toradioisotopes such as ¹²⁵I; enzyme labelling systems that generate adetectable calorimetric signal or light when exposed to substrate; andfluorescent labels.

[0172] Where recombinant DNA technology is used to produce thedifferentially expressed or pathway gene protein for such assay systems,it may be advantageous to engineer fusion proteins that can facilitatelabeling, immobilization and/or detection.

[0173] Indirect labeling involves the use of a protein, such as alabeled antibody, which specifically binds to either a differentiallyexpressed or pathway gene product. Such antibodies include but are notlimited to polyclonal, monoclonal, chimeric, single chain, Fab fragmentsand fragments-produced by an Fab expression library.

[0174] 5.4.3. Differentially Expressed or Pathway Gene ProductAntibodies

[0175] Described herein are methods for the production of antibodiescapable of specifically recognizing one or more differentially expressedor pathway gene epitopes. Such antibodies may include, but are notlimited to polyclonal antibodies, monoclonal antibodies (mAbs),humanized or chimeric antibodies, single chain antibodies, Fabfragments, F(ab′)₂ fragments, fragments produced by a Fab expressionlibrary, anti-idiotypic (anti-Id) antibodies, and epitope-bindingfragments of any of the above. Such antibodies may be used, for example,in the detection of a fingerprint, target, or pathway gene in abiological sample, or, alternatively, as a method for the inhibition ofabnormal target gene activity. Thus, such antibodies may be utilized aspart of cardiovascular disease treatment methods, and/or may be used aspart of diagnostic techniques whereby patients may be tested forabnormal levels of fingerprint, target, or pathway gene proteins, or forthe presence of abnormal forms of the such proteins.

[0176] For the production of antibodies to a differentially expressed orpathway gene, various host animals may be immunized by injection with adifferentially expressed or pathway gene protein, or a portion thereof.Such host animals may include but are not limited to rabbits, mice, andrats, to name but a few. Various adjuvants may be used to increase theimmunological response, depending on the host species, including but notlimited to Freund's (complete and incomplete), mineral gels such asaluminum hydroxide, surface active substances such as lysolecithin,pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpethemocyanin, dinitrophenol, and potentially useful human adjuvants suchas BCG (bacille Calmette-Guerin) and Corynebacterium parvum.

[0177] Polyclonal antibodies are heterogeneous populations of antibodymolecules derived from the sera of animals immunized with an antigen,such as target gene product, or an antigenic functional derivativethereof. For the production of polyclonal antibodies, host animals suchas those described above, may be immunized by injection withdifferentially expressed or pathway gene product supplemented withadjuvants as also described above.

[0178] Monoclonal antibodies, which are homogeneous populations ofantibodies to a particular antigen, may be obtained by any techniquewhich provides for the production of antibody molecules by continuouscell lines in culture. These include, but are not limited to thehybridoma technique of Kohler and Milstein, (1975, Nature 256:495-497;and U.S. Pat. No. 4,376,110), the human B-cell hybridoma technique(Kosbor et al., 1983, Immunology Today 4:72; Cole et al., 1983, Proc.Natl. Acad. Sci. USA 80:2026-2030), and the EBV-hybridoma technique(Cole et al., 1985, Monoclonal Antibodies And Cancer Therapy, Alan R.Liss, Inc., pp. 77-96). Such antibodies may be of any immunoglobulinclass including IgG, IgM, IgE, IgA, IgD and any subclass thereof. Thehybridoma producing the mAb of this invention may be cultivated in vitroor in vivo. Production of high titers of mabs in vivo makes this thepresently preferred method of production.

[0179] In addition, techniques developed for the production of “chimericantibodies” (Morrison et al., 1984, Proc. Natl. Acad. Sci.,81:6851-6855; Neuberger et al., 1984,Nature, 312:604-608; Takeda et al.,1985, Nature, 314:452-454) by splicing the genes from a mouse antibodymolecule of appropriate antigen specificity together with genes from ahuman antibody molecule of appropriate biological activity can be used.A chimeric antibody is a molecule in which different portions arederived from different animal species, such as those having a variableregion derived from a murine mAb and a human immunoglobulin constantregion.

[0180] Alternatively, techniques described for the production of singlechain antibodies (U.S. Pat. No. 4,946,778;Bird, 1988, Science242:423-426; Huston et al., 1988, Proc.

[0181] Natl. Acad. Sci. USA 85:5879-5883; and Ward et al., 1989, Nature334:544-546) can be adapted to produce differentially expressed orpathway gene-single chain antibodies. Single chain antibodies are formedby linking the heavy and light chain fragments of the Fv region via anamino acid bridge, resulting in a single chain polypeptide.

[0182] Antibody fragments which recognize specific epitopes may begenerated by known techniques. For example, such fragments include butare not limited to: the F(ab′)₂ fragments which can be produced bypepsin digestion of the antibody molecule and the Fab fragments whichcan be generated by reducing the disulfide bridges of the F(ab′)₂fragments. Alternatively, Fab expression libraries may be constructed(Huse et al., 1989, Science, 246:1275-1281) to allow rapid and easyidentification of monoclonal Fab fragments with the desired specificity.

[0183] 5.4.4. Cell- and Animal-Based Model Systems

[0184] Described herein are cell- and animal-based systems which act asmodels for cardiovascular disease. These systems may be used in avariety of applications. For example, the cell- and animal-based modelsystems may be used to further characterize differentially expressed andpathway genes, as described, above, in Section 5.3. Such furthercharacterization may, for example, indicate that a differentiallyexpressed gene is a target gene. Second, such assays may be utilized aspart of screening strategies designed to identify compounds which arecapable of ameliorating cardiovascular disease symptoms, as described,below, in Section 5.5.4. Thus, the animal- and cell-based models may beused to identify drugs, pharmaceuticals, therapies and interventionswhich may be effective in treating cardiovascular disease. In addition,as described in detail, below, in Section 5.7.1, such animal models maybe used to determine the LD₅₀, and the ED₅₀ in animal subjects, and suchdata can be used to determine the in vivo efficacy of potentialcardiovascular disease treatments.

[0185] 5.4.4.1. Animal-Based Systems

[0186] Animal-based model systems of cardiovascular disease may include,but are not limited to, non-recombinant and engineered transgenicanimals.

[0187] Non-recombinant animal models for cardiovascular disease mayinclude, for example, genetic models. Such genetic cardiovasculardisease models may include, for example, apoB or apoR deficient pigs(Rapacz, et al., 1986, Science 234:1573-1577) and Watanabe heritablehyperlipidemic (WHHL) rabbits (Kita et al., 1987, Proc. Natl. Acad. SciUSA 84: 5928-5931).

[0188] Non-recombinant, non-genetic animal models of atherosclerosis mayinclude, for example, pig, rabbit, or rat models in which the animal hasbeen exposed to either chemical wounding through dietary supplementationof LDL, or mechanical wounding through balloon catheter angioplasty, forexample.

[0189] Additionally, animal models exhibiting cardiovascular diseasesymptoms may be engineered by utilizing, for example, target genesequences such as those described, above, in Section 5.4.1, inconjunction with techniques for producing transgenic animals that arewell known to those of skill in the art. For example, target genesequences may be introduced into, and overexpressed in, the genome ofthe animal of interest, or, if endogenous target gene sequences arepresent, they may either be overexpressed or, alternatively, bedisrupted in order to underexpress or inactivate target gene expression,such as described for the disruption of apoE in mice (Plump et al.,1992, Cell 71: 343-353).

[0190] In order to overexpress a target gene sequence, the codingportion of the target gene sequence may be ligated to a regulatorysequence which is capable of driving gene expression in the animal andcell type of interest. Such regulatory regions will be well known tothose of skill in the art, and may be utilized in the absence of undueexperimentation.

[0191] The use of such a genetically engineered animal-based system isdescribed in detail in the example provided in Section 7, below, for thetarget gene bcl-2 (see Table 1, in Section 5.4.1, above). Briefly, bcl-2expression first was shown to be down-regulated in the apoE-deficientatherosclerosis mouse model. Then, a transgenic mouse was engineeredbearing the human bcl-2 gene under the control of a promoter which isinduced under atherogenic conditions. To test the effect of theinduction of bcl-2 under such conditions, the transgenic mouse iscrossed with the apoE-deficient mouse. apoE-deficient progeny bearingthe highly expressible bcl-2 gene are then examined for plaque formationand development. Reduction in plaque formation and development in theseprogeny confirms the effectiveness of intervening in cardiovasculardisease through this target gene.

[0192] For underexpression of an endogenous target gene sequence, such asequence may be isolated and engineered such that when reintroduced intothe genome of the animal of interest, the endogenous target gene alleleswill be inactivated. Preferably, the engineered target gene sequence isintroduced via gene targeting such that the endogenous target sequenceis disrupted upon integration of the engineered target gene sequenceinto the animal's genome. Gene targeting is discussed, below, in thisSection.

[0193] Animals of any species, including, but not limited to, mice,rats, rabbits, guinea pigs, pigs, micro-pigs, goats, and non-humanprimates, e.g., baboons, monkeys, and chimpanzees may be used togenerate cardiovascular disease animal models.

[0194] Any technique known in the art may be used to introduce a targetgene transgene into animals to produce the founder lines of transgenicanimals. Such techniques include, but are not limited to pronuclearmicroinjection (Hoppe, P. C. and Wagner, T. E., 1989, U.S. Pat. No.4,873,191); retrovirus mediated gene transfer into germ lines (Van derPutten et al., 1985, Proc. Natl. Acad. Sci., USA 82:6148-6152); genetargeting in embryonic stem cells (Thompson et al., 1989, Cell56:313-321); electroporation of embryos (Lo, 1983, Mol Cell. Biol.3:1803-1814); and sperm-mediated gene transfer (Lavitrano et al., 1989,Cell 57:717-723); etc. For a review of such techniques, see Gordon,1989, Transgenic Animals, Intl. Rev. Cytol. 115:171-229, which isincorporated by reference herein in its entirety.

[0195] The present invention provides for transgenic animals that carrythe transgene in all their cells, as well as animals which carry thetransgene in some, but not all their cells, i.e., mosaic animals. Thetransgene may be integrated as a single transgene or in concatamers,e.g., head-to-head tandems or head-to-tail tandems. The transgene mayalso be selectively introduced into and activated in a particular celltype by following, for example, the teaching of Lasko et al. (Lasko, M.et al., 1992, Proc. Natl. Acad. Sci. USA 89: 6232-6236). The regulatorysequences required for such a cell-type specific activation will dependupon the particular cell type of interest, and will be apparent to thoseof skill in the art. When it is desired that the target gene transgenebe integrated into the chromosomal site of the endogenous target gene,gene targeting is preferred. Briefly, when such a technique is to beutilized, vectors containing some nucleotide sequences homologous to theendogenous target gene of interest are designed for the purpose ofintegrating, via homologous recombination with chromosomal sequences,into and disrupting the function of the nucleotide sequence of theendogenous target gene. The transgene may also be selectively introducedinto a particular cell type, thus inactivating the endogenous gene ofinterest in only that cell type, by following, for example, the teachingof Gu et al. (Gu, et al., 1994, Science 265: 103-106). The regulatorysequences required for such a cell-type specific inactivation willdepend upon the particular cell type of interest, and will be apparentto those of skill in the art.

[0196] Once transgenic animals have been generated, the expression ofthe recombinant target gene and protein may be assayed utilizingstandard techniques. Initial screening may be accomplished by Southernblot analysis or PCR techniques to analyze animal tissues to assaywhether integration of the transgene has taken place. The level of mRNAexpression of the transgene in the tissues of the transgenic animals mayalso be assessed using techniques which include but are not limited toNorthern blot analysis of tissue samples obtained from the animal, insitu hybridization analysis, and RT-PCR. Samples of targetgene-expressing tissue, may also be evaluated immunocytochemically usingantibodies specific for the target gene transgene gene product ofinterest.

[0197] The target gene transgenic animals that express target gene mRNAor target gene transgene peptide (detected immunocytochemically, usingantibodies directed against the target gene product's epitopes) ateasily detectable levels should then be further evaluated to identifythose animals which display characteristic cardiovascular diseasesymptoms. Such symptoms may include, for example, increased prevalenceand size of fatty streaks and/or cardiovascular disease plaques.

[0198] Additionally, specific cell types within the transgenic animalsmay be analyzed and assayed for cellular phenotypes characteristic ofcardiovascular disease. In the case of monocytes, such phenotypes mayinclude but are not limited to increases in rates of LDL uptake,adhesion to endothelial cells, transmigration, foam cell formation,fatty streak formation, and production of foam cell specific products.Cellular phenotype assays are discussed in detail in Section 5.4.4.2,below. Further, such cellular phenotypes may include a particular celltype's fingerprint pattern of expression as compared to knownfingerprint expression profiles of the particular cell type in animalsexhibiting cardiovascular disease symptoms. Fingerprint profiles aredescribed in detail in Section 5.8.1, below. Such transgenic animalsserve as suitable model systems for cardiovascular disease.

[0199] Once target gene transgenic founder animals are produced, theymay be bred, inbred, outbred, or crossbred to produce colonies of theparticular animal. Examples of such breeding strategies include but arenot limited to: outbreeding of founder animals with more than oneintegration site in order to establish separate lines; inbreeding ofseparate lines in order to produce compound target gene transgenics thatexpress the target gene transgene of interest at higher levels becauseof the effects of additive expression of each target gene transgene;crossing of heterozygous transgenic animals to produce animalshomozygous for a given integration site in order both to augmentexpression and eliminate the possible need for screening of animals byDNA analysis; crossing of separate homozygous lines to produce compoundheterozygous or homozygous lines; breeding animals to different inbredgenetic backgrounds so as to examine effects of modifying alleles onexpression of the target gene transgene and the development ofcardiovascular disease symptoms. one such approach is to cross thetarget gene transgenic founder animals with a wild type strain toproduce an Fl generation that exhibits cardiovascular disease symptoms.The Fl generation may then be inbred in order to develop a homozygousline, if it is found that homozygous target gene transgenic animals areviable.

[0200] 5.4.4.2. Cell-Based Assays

[0201] Cells that contain and express target gene sequences which encodetarget gene protein, and, further, exhibit cellular phenotypesassociated with cardiovascular disease, may be utilized to identifycompounds that exhibit anti-cardiovascular disease activity. In the caseof monocytes, such phenotypes may include but are not limited toincreases in rates of LDL uptake, adhesion to endothelial cells,transmigration, foam cell formation, fatty streak formation, andproduction by foam cells of growth factors such as bFGF, IGF-I, VEGF,IL-1, M-CSF, TGFβ, TGFα, TNFA, HB-EGF, PDGF, IFN-γ, and GM-CSF.Transmigration rates, for example, may be measured using the in vitrosystem of Navab et al., described in Section 5.1.1.3, above, byquantifying the number of monocytes that migrate across the endothelialmonolayer and into the collagen layer of the subendothelial space.

[0202] Such cells may include non-recombinant cell lines, such as U937(ATCC# CRL1593) and THP-1 (TIB202). Further, such cells may includerecombinant, transgenic cell lines. For example, the cardiovasculardisease animal models of the invention, discussed, above, in Section5.4.4.1, may be used to generate cell lines, containing one or more celltypes involved in cardiovascular disease, that can be used as cellculture models for this disorder. While primary cultures derived fromthe cardiovascular disease transgenic animals of the invention may beutilized, the generation of continuous cell lines is preferred. Forexamples of techniques which may be used to derive a continuous cellline from the transgenic animals, see Small et al., 1985, Mol. CellBiol. 5:642-648.

[0203] Alternatively, cells of a cell type known to be involved incardiovascular disease may be transfected with sequences capable ofincreasing or decreasing the amount of target gene expression within thecell. For example, target gene sequences may be introduced into, andoverexpressed in, the genome of the cell of interest, or, if endogenoustarget gene sequences are present, they may be either overexpressed or,alternatively disrupted in order to underexpress or inactivate targetgene expression.

[0204] In order to overexpress a target gene sequence, the codingportion of the target gene sequence may be ligated to a regulatorysequence which is capable of driving gene expression in the cell type ofinterest. Such regulatory regions will be well known to those of skillin the art, and may be utilized in the absence of undue experimentation.

[0205] For underexpression of an endogenous target gene sequence, such asequence may be isolated and engineered such that when reintroduced intothe genome of the cell type of interest, the endogenous target genealleles will be inactivated. Preferably, the engineered target genesequence is introduced via gene targeting such that the endogenoustarget sequence is disrupted upon integration of the engineered targetgene sequence into the cell's genome. Target gene introduction isdiscussed, above, in Section 5.4.4.1.

[0206] Transfection of target gene sequence nucleic acid may beaccomplished by utilizing standard techniques See, for example, Ausubel,1989, supra. Transfected cells should be evaluated for the presence ofthe recombinant target gene sequences, for expression and accumulationof target gene mRNA, and for the presence of recombinant target geneprotein production. In instances wherein a decrease in target geneexpression is desired, standard techniques may be used to demonstratewhether a decrease in endogenous target gene expression and/or in targetgene product production is achieved.

[0207] 5.5. Screening Assays for Compounds that Interact with the TargetGene Product

[0208] The following assays are designed to identify compounds that bindto target gene products, bind to other cellular or extracellularproteins that interact with a target gene product, and interfere withthe interaction of the target gene product with other cellular orextracellular proteins. For example, in the case of the rchd523 geneproduct, which is a transmembrane receptor-type protein, such techniquescan identify ligands for such a receptor. An rchd523 gene product ligandcan, for example, act as the basis for amelioration of suchcardiovascular diseases as atherosclerosis, ischemia/reperfusion,hypertension, restenosis, and arterial inflammation, given that rchd523up-regulation is specific to endothelial cells. Such compounds mayinclude, but are not limited to peptides, antibodies, or small organicor inorganic compounds. Methods for the identification of such compoundsare described in Section 5.5.1, below. Such compounds may also includeother cellular proteins. Methods for the identification of such cellularproteins are described, below, in Section 5.5.2.

[0209] Compounds identified via assays such as those described hereinmay be useful, for example, in elaborating the biological function ofthe target gene product, and for ameliorating cardiovascular disease. Ininstances whereby a cardiovascular disease condition results from anoverall lower level of target gene expression and/or target gene productin a cell or tissue, compounds that interact with the target geneproduct may include compounds which accentuate or amplify the activityof the bound target gene protein. Such compounds would bring about aneffective increase in the level of target gene product activity, thusameliorating symptoms.

[0210] In other instances mutations within the target gene may causeaberrant types or excessive amounts of target gene proteins to be madewhich have a deleterious effect that leads to cardiovascular disease.Similarly, physiological conditions may cause an excessive increase intarget gene expression leading to cardiovascular disease. In such cases,compounds that bind target gene protein may be identified that inhibitthe activity of the bound target gene protein. Assays for testing theeffectiveness of compounds, identified by, for example, techniques suchas those described in this Section are discussed, below, in Section5.5.4.

[0211] 5.5.1. in vitro Screening Assays for Compounds that Bind to theTarget Gene Product

[0212] In vitro systems may be designed to identify compounds capable ofbinding the target gene of the invention. Such compounds may include,but are not limited to, peptides made of D-and/or L-configuration aminoacids (in, for example, the form of random peptide libraries; see e.g.,Lam, K. S. et al., 1991, Nature 354:82-84), phosphopeptides (in, forexample, the form of random or partially degenerate, directedphosphopeptide libraries; see, e.g., Songyang, Z. et al., 1993, Cell72:767-778), antibodies, and small organic or inorganic molecules.Compounds identified may be useful, for example, in modulating theactivity of target gene proteins, preferably mutant target geneproteins, may be useful in elaborating the biological function of thetarget gene protein, may be utilized in screens for identifyingcompounds that disrupt normal target gene interactions, or may inthemselves disrupt such interactions.

[0213] The principle of the assays used to identify compounds that bindto the target gene protein involves preparing a reaction mixture of thetarget gene protein and the test compound under conditions and for atime sufficient to allow the two components to interact and bind, thusforming a complex which can be removed and/or detected in the reactionmixture. These assays can be conducted in a variety of ways. Forexample, one method to conduct such an assay would involve anchoring thetarget gene or the test substance onto a solid phase and detectingtarget gene/test substance complexes anchored on the solid phase at theend of the reaction. In one embodiment of such a method, the target geneprotein may be anchored onto a solid surface, and the test compound,which is not anchored, may be labeled, either directly or indirectly.

[0214] In practice, microtitre plates are conveniently utilized. Theanchored component may be immobilized by non-covalent or covalentattachments. Non-covalent attachment may be accomplished simply bycoating the solid surface with a solution of the protein and drying.Alternatively, an immobilized antibody, preferably a monoclonalantibody, specific for the protein may be used to anchor the protein tothe solid surface. The surfaces may be prepared in advance and stored.

[0215] In order to conduct the assay, the nonimmobilized component isadded to the coated surface containing the anchored component. After thereaction is complete, unreacted components are removed (e.g., bywashing) under conditions such that any complexes formed will remainimmobilized on the solid surface. The detection of complexes anchored onthe solid surface can be accomplished in a number of ways. Where thepreviously nonimmobilized component is pre-labeled, the detection oflabel immobilized on the surface indicates that complexes were formed.Where the previously nonimmobilized component is not pre-labeled, anindirect label can be used to detect complexes anchored on the surface;e.g., using a labeled antibody specific for the previouslynonimmobilized component (the antibody, in turn, may be directly labeledor indirectly labeled with a labeled anti-Ig antibody).

[0216] Alternatively, a reaction can be conducted in a liquid phase, thereaction products separated from unreacted components, and complexesdetected; e.g., using an immobilized antibody specific for target geneproduct or the test compound to anchor any complexes formed in solution,and a labeled antibody specific for the other component of the possiblecomplex to detect anchored complexes.

[0217] Compounds that are shown to bind to a particular target geneproduct through one of the methods described above can be further testedfor their ability to elicit a biochemical response from the target geneprotein. A particular embodiment is described herein for receptorproteins involved in signal transduction, including but not limited tothe rchd523 gene product. Compounds that interact with a target geneproduct receptor domain, can be screened for their ability to functionas ligands, i.e., to bind to the receptor protein in a manner thattriggers the signal transduction pathway. Useful receptor fragments oranalogs in the invention are those which interact with ligand. Thereceptor component can be assayed functionally, i.e., for its ability tobind ligand and mobilize Ca⁺⁺ (see below). These assays include, ascomponents, ligand and a recombinant target gene product (or a suitablefragment or analog) configured to permit detection of binding.

[0218] For example, and not by way of limitation, a recombinant receptormay be used to screen for ligands by its ability to mediateligand-dependent mobilization of calcium. Cells, preferably myelomacells or Xenopus oocytes, transfected with a target gene expressionvector (constructed according to the methods described in Section 5.4.2,above) are loaded with FURA-2 or INDO-1 by standard techniques.Mobilization of Ca²⁺ induced by ligand is measured by fluorescencespectroscopy as previously described (Grynkiewicz et al., 1985, J. Biol.Chem. 260:3440). Ligands that react with the target gene productreceptor domain, therefore, can be identified by their ability toproduce a fluorescent signal. Their receptor binding activities can bequantified and compared by measuring the level of fluorescence producedover background.

[0219] The rchd523 gene product consists of a G protein-coupled receptorwith multiple transmembrane domains. The Ca²+mobilization assay,therefore, can be used to screen compounds that are ligands of therchd523 receptor. This screening method is described in detail withrespect to rchd523 in the example in Section 12, below. Identificationof rchd523 ligand, and measuring the activity of the ligand-receptorcomplex, leads to the identification of antagonists of this interaction,as described in Section 5.5.3, below. Such antagonists are useful in thetreatment of cardiovascular disease.

[0220] 5.5.2. Assays for Cellular or Extracellular Proteins thatInteract with the Target Gene Product

[0221] Any method suitable for detecting protein-protein interactionsmay be employed for identifying novel target protein-cellular orextracellular protein interactions. These methods are outlined inSection 5.2., supra, for the identification of pathway genes, and may beutilized herein with respect to the identification of proteins whichinteract with identified target proteins. In such a case, the targetgene serves as the known “bait” gene.

[0222] 5.5.3. Assays for Compounds that Interfere with InteractionBetween Target Gene Product and Other Compounds

[0223] The target gene proteins of the invention may, in vivo, interactwith one or more cellular or extracellular proteins. Such proteins mayinclude, but are not limited to, those proteins identified via methodssuch as those described, above, in Section 5.5.2. For the purposes ofthis discussion, target gene products and such cellular andextracellular proteins are referred to herein as “binding partners”.Compounds that disrupt such interactions may be useful in regulating theactivity of the target gene proteins, especially mutant target geneproteins. Such compounds may include, but are not limited to moleculessuch as antibodies, peptides, and the like described in Section 5.5.1.above.

[0224] The basic principle of the assay systems used to identifycompounds that interfere with the interaction between the target geneprotein, and its cellular or extracellular protein binding partner orpartners involves preparing a reaction mixture containing the targetgene protein and the binding partner under conditions and for a timesufficient to allow the two proteins to interact and bind, thus forminga complex. In order to test a compound for inhibitory activity, thereaction mixture is prepared in the presence and absence of the testcompound. The test compound may be initially included in the reactionmixture or may be added at a time subsequent to the addition of targetgene and its cellular or extracellular binding partner. Control reactionmixtures are incubated without the test compound or with a placebo. Theformation of any complexes between the target gene protein and thecellular or extracellular binding partner is then detected. Theformation of a complex in the control reaction, but not in the reactionmixture containing the test compound, indicates that the compoundinterferes with the interaction of the target gene protein and theinteractive binding partner protein. Additionally, complex formationwithin reaction mixtures containing the test compound and a normaltarget gene protein may also be compared to complex formation withinreaction mixtures containing the test compound and mutant target geneprotein. This comparison may be important in those cases wherein it isdesirable to identify compounds that disrupt interactions of mutant butnot normal target gene proteins.

[0225] The assay for compounds that interfere with the interaction ofthe binding partners can be conducted in a heterogeneous or homogeneousformat. Heterogeneous assays involve anchoring one of the bindingpartners onto a solid phase and detecting complexes anchored on thesolid phase at the end of the reaction. In homogeneous assays, theentire reaction is carried out in a liquid phase. In either approach,the order of addition of reactants can be varied to obtain differentinformation about the compounds being tested. For example, testcompounds that interfere with the interaction between the bindingpartners, e.g., by competition, can be identified by conducting thereaction in the presence of the test substance; i.e., by adding the testsubstance to the reaction mixture prior to or simultaneously with thetarget gene protein and interactive cellular or extracellular protein.Alternatively, test compounds that disrupt preformed complexes, e.g.compounds with higher binding constants that displace one of the bindingpartners from the complex, can be tested by adding the test compound tothe reaction mixture after complexes have been formed. The variousformats are described briefly below.

[0226] In a heterogeneous assay system, either the target gene proteinor the interactive cellular or extracellular binding partner protein, isanchored onto a solid surface, and its binding partner, which is notanchored, is labeled, either directly or indirectly. In practice,microtitre plates are conveniently utilized. The anchored species may beimmobilized by non-covalent or covalent attachments. Non-covalentattachment may be accomplished simply by coating the solid surface witha solution of the protein and drying. Alternatively, an immobilizedantibody specific for the protein may be used to anchor the protein tothe solid surface. The surfaces may be prepared in advance and stored.

[0227] In order to conduct the assay, the binding partner of theimmobilized species is exposed to the coated surface with or without thetest compound. After the reaction is complete, unreacted components areremoved (e.g., by washing) and any complexes formed will remainimmobilized on the solid surface. The detection of complexes anchored onthe solid surface can be accomplished in a number of ways. Where thebinding partner was pre-labeled, the detection of label immobilized onthe surface indicates that complexes were formed. Where the bindingpartner is not pre-labeled, an indirect label can be used to detectcomplexes anchored on the surface; e.g., using a labeled antibodyspecific for the binding partner (the antibody, in turn, may be directlylabeled or indirectly labeled with a labeled anti-Ig antibody).Depending upon the order of addition of reaction components, testcompounds which inhibit complex formation or which disrupt preformedcomplexes can be detected.

[0228] Alternatively, the reaction can be conducted in a liquid phase inthe presence or absence of the test compound, the reaction productsseparated from unreacted components, and complexes detected; e.g., usingan immobilized antibody specific for one binding partner to anchor anycomplexes formed in solution, and a labeled antibody specific for theother binding partner to detect anchored complexes. Again, dependingupon the order of addition of reactants to the liquid phase, testcompounds which inhibit complex or which disrupt preformed complexes canbe identified.

[0229] In an alternate embodiment of the invention, a homogeneous assaycan be used. In this approach, a preformed complex of the target geneprotein and the interactive cellular or extracellular protein isprepared in which one of the binding partners is labeled, but the signalgenerated by the label is quenched due to complex formation (see, e.g.,U.S. Pat. No. 4,109,496 by Rubenstein which utilizes this approach forimmunoassays). The addition of a test substance that competes with anddisplaces one of the binding partners from the preformed complex willresult in the generation of a signal above background. In this way, testsubstances which disrupt target gene protein-cellular or extracellularprotein interaction can be identified.

[0230] In a particular embodiment, the target gene protein can beprepared for immobilization using recombinant DNA techniques describedin Section 5.4.2, supra. For example, the target gene coding region canbe fused to a glutathione-S-transferase (GST) gene, using a fusionvector such as PGEX-5X-1, in such a manner that its binding activity ismaintained in the resulting fusion protein. The interactive cellular orextracellular protein can be purified and used to raise a monoclonalantibody, using methods routinely practiced in the art and describedabove, in Section 5.4.3. This antibody can be labeled with theradioactive isotope ¹²⁵I, for example, by methods routinely practiced inthe art. In a heterogeneous assay, e.g., the GST-target gene fusionprotein can be anchored to glutathione-agarose beads. The interactivecellular or extracellular binding partner protein can then be added inthe presence or absence of the test compound in a manner that allowsinteraction and binding to occur. At the end of the reaction period,unbound material can be washed away, and the labeled monoclonal antibodycan be added to the system and allowed to bind to the complexed bindingpartners. The interaction between the target gene protein and theinteractive cellular or extracellular binding partner protein can bedetected by measuring the amount of radioactivity that remainsassociated with the glutathione-agarose beads. A successful inhibitionof the interaction by the test compound will result in a decrease inmeasured radioactivity.

[0231] Alternatively, the GST-target gene fusion protein and theinteractive cellular or extracellular binding partner protein can bemixed together in liquid in the absence of the solid glutathione-agarosebeads. The test compound can be added either during or after the bindingpartners are allowed to interact. This mixture can then be added to theglutathione-agarose beads and unbound material is washed away. Again theextent of inhibition of the binding partner interaction can be detectedby adding the labeled antibody and measuring the radioactivityassociated with the beads.

[0232] In another embodiment of the invention, these same techniques canbe employed using peptide fragments that correspond to the bindingdomains of the target gene protein and the interactive cellular orextracellular protein, respectively, in place of one or both of the fulllength proteins. Any number of methods routinely practiced in the artcan be used to identify and isolate the protein's binding site. Thesemethods include, but are not limited to, mutagenesis of one of the genesencoding the proteins and screening for disruption of binding in aco-immunoprecipitation assay. Compensating mutations in the target genecan be selected. Sequence analysis of the genes encoding the respectiveproteins will reveal the mutations that correspond to the region of theprotein involved in interactive binding. Alternatively, one protein canbe anchored to a solid surface using methods described in this Sectionabove, and allowed to interact with and bind to its labeled bindingpartner, which has been treated with a proteolytic enzyme, such astrypsin. After washing, a short, labeled peptide comprising the bindingdomain may remain associated with the solid material, which can beisolated and identified by amino acid sequencing. Also, once the genecoding for the for the cellular or extracellular protein is obtained,short gene segments can be engineered to express peptide fragments ofthe protein, which can then be tested for binding activity and purifiedor synthesized.

[0233] For example, and not by way of limitation, target gene can beanchored to a solid material as described above in this Section bymaking a GST-target gene fusion protein and allowing it to bind toglutathione agarose beads. The interactive cellular or extracellularbinding partner protein can be labeled with a radioactive isotope, suchas ³⁵S, and cleaved with a proteolytic enzyme such as trypsin. Cleavageproducts can then be added to the anchored GST-target gene fusionprotein and allowed to bind. After washing away unbound peptides,labeled bound material, representing the cellular or extracellularbinding partner protein binding domain, can be eluted, purified, andanalyzed for amino acid sequence by techniques well known in the art;e.g., using the Edman degradation procedure (see e.g., Creighton, 1983,Proteins: Structures and Molecular Principles, W. H. Freeman & Co.,N.Y., pp. 34-49). Peptides so identified can be produced, usingtechniques well known in the art, either synthetically (see e.g.,Creighton, 1983, supra at pp. 50-60) or, if the gene has already beenisolated, by using recombinant DNA technology, as described in Section5.4.2, supra.

[0234] A particular embodiment of the invention features a method ofscreening candidate compounds for their ability to antagonize theinteraction between ligand and the receptor domain of a target geneproduct, including but not limited to the receptor domain of the rchd523gene product. The rchd523 gene product, which is a G protein-coupledreceptor protein containing multiple transmembrane domains, isespecially useful in screening for antagonists of ligand-receptorinteractions. The method involves: a) mixing a candidate antagonistcompound with a first compound which includes a recombinant target geneproduct comprising a receptor domain (or ligand-binding fragment oranalog) on the one hand and with a second compound which includes ligandon the other hand; b) determining whether the first and second compoundsbind; and c) identifying antagonistic compounds as those which interferewith the binding of the first compound to the second compound and/orwhich reduce the ligand-mediated release of intracellular Ca⁺⁺.

[0235] By an “antagonist” is meant a molecule which inhibits aparticular activity, in this case, the ability of ligand to interactwith a target gene product receptor domain and/or to trigger thebiological events resulting from such an interaction (e.g., release ofintracellular Ca⁺⁺). Preferred therapeutics include antagonists, e.g.,peptide fragments (particularly, fragments derived from the N-terminalextracellular domain), antibodies (particularly, antibodies whichrecognize and bind the N-terminal extracellular domain), or drugs, whichblock ligand or target gene product function by interfering with theligand-receptor interaction.

[0236] Because the receptor component of the target gene product can beproduced by recombinant techniques and because candidate antagonists maybe screened in vitro, the instant invention provides a simple and rapidapproach to the identification of useful therapeutics.

[0237] Specific receptor fragments of interest include any portions ofthe target gene products that are capable of interaction with ligand,for example, all or part of the N-terminal extracellular domain. Suchportions include the transmembrane segments and portions of the receptordeduced to be extracellular. Such fragments may be useful as antagonists(as described above), and are also useful as immunogens for producingantibodies which neutralize the activity of the target gene product invivo (e.g., by interfering with the interaction between the receptor andligand; see below). Extracellular regions may be identified bycomparison with related proteins of similar structure (e.g., othermembers of the G-protein-coupled receptor superfamily); useful regionsare those exhibiting homology to the extracellular domains ofwell-characterized members of the family.

[0238] Alternatively, from the primary amino acid sequence, thesecondary protein structure and, therefore, the extracellular domainregions may be deduced semi-empirically using ahydrophobicity/hydrophilicity calculation such as the Chou-Fasman method(see, e.g., Chou and Fasman, Ann. Rev. Biochem. 47:251, 1978).Hydrophilic domains, particularly ones surrounded by hydrophobicstretches (e.g., transmembrane domains) present themselves as strongcandidates for extracellular domains. Finally, extracellular domains maybe identified experimentally using standard enzymatic digest analysis,e.g., tryptic digest analysis.

[0239] Candidate fragments (e.g., all or part of the transmembranesegments or any extracellular fragment) are tested for interaction withligand by the assays described herein (e.g., the assay described above).Such fragments are also tested for their ability to antagonize theinteraction between ligand and its endogenous receptor using the assaysdescribed herein. Analogs of useful receptor fragments (as describedabove) may also be produced and tested for efficacy as screeningcomponents or antagonists (using the assays described herein); suchanalogs are also considered to be useful in the invention.

[0240] Of particular interest are receptor fragments encompassing theextracellular main-terminal domain (or a ligand binding fragmentthereof). Also of interest are the target gene product extracellularloops. Peptide fragments derived from these extracellular loops may alsobe used as antagonists, particularly if the loops cooperate with theamino-terminal domain to facilitate ligand binding. Alternatively, suchloops and extracellular N-terminal domain (as well as the full lengthtarget gene product) provide immunogens for producing anti-target geneproduct antibodies.

[0241] Binding of ligand to its receptor may be assayed by any of themethods described above in Section 5.5.1. Preferably, cells expressingrecombinant target gene product (or a suitable target gene productfragment or analog) are immobilized on a solid substrate (e.g., the wallof a microtitre-plate or a column) and reacted with detectably-labelledligand (as described above). Binding is assayed by the detection labelin association with the receptor component (and, therefore, inassociation with the solid substrate). Binding of labelled ligand toreceptor-bearing cells is used as a “control” against which antagonistassays are measured. The antagonist assays involve incubation of thetarget gene product-bearing cells with an appropriate amount ofcandidate antagonist. To this mix, an equivalent amount to labelledligand is added. An antagonist useful in the invention specificallyinterferes with labelled ligand binding to the immobilizedreceptor-expressing cells.

[0242] An antagonist is then tested for its ability to interfere withligand function, i.e., to specifically interfere with labelled ligandbinding without resulting in signal transduction normally mediated bythe receptor. To test this using a functional assay, stably transfectedcell lines containing the target gene product can be produced asdescribed herein and reporter compounds such as the calcium bindingagent, FURA-2, loaded into the cytoplasm by standard techniques.Stimulation of the heterologous target gene product with ligand oranother agonist leads to intracellular calcium release and theconcomitant fluorescence of the calcium-FURA-2 complex. This provides aconvenient means for measuring agonist activity. Inclusion of potentialantagonists along with ligand allows for the screening andidentification of authentic receptor antagonists as those whicheffectively block ligand binding without producing fluorescence (i.e.,without causing the mobilization of intracellular Ca⁺⁺). Such anantagonist may be expected to be a useful therapeutic agent forcardiovascular disorders.

[0243] Appropriate candidate antagonists include target gene productfragments, particularly fragments containing a ligand-binding portionadjacent to or including one or more transmembrane segments or anextracellular domain of the receptor (described above); such fragmentswould preferably including five or more amino acids. Other candidateantagonists include analogs of ligand and other peptides as well asnon-peptide compounds and anti-target gene product antibodies designedor derived from analysis of the receptor.

[0244] This screening method is described in detail with respect to therchd523 gene in the example in Section 12, below. Because the rchd523gene product is a G protein-coupled receptor, antagonists of theinteraction between the rchd523 gene product and its natural ligandprovide excellent candidates for compounds effective in the treatment ofcardiovascular disease.

[0245] 5.5.4. Assays for Amelioration of Cardiovascular Disease Symptoms

[0246] Any of the binding compounds, including but not limited tocompounds such as those identified in the foregoing assay systems, maybe tested for the ability to ameliorate cardiovascular disease symptoms.Cell-based and animal model-based assays for the identification ofcompounds exhibiting such an ability to ameliorate cardiovasculardisease symptoms are described below.

[0247] First, cell-based systems such as those described, above, inSection 5.4.4.2., may be used to identify compounds which may act toameliorate cardiovascular disease symptoms. For example, such cellsystems may be exposed to a compound, suspected of exhibiting an abilityto ameliorate cardiovascular disease symptoms, at a sufficientconcentration and for a time sufficient to elicit such an ameliorationof cardiovascular disease symptoms in the exposed cells. After exposure,the cells are examined to determine whether one or more of thecardiovascular disease cellular phenotypes has been altered to resemblea more normal or more wild type, non-cardiovascular disease phenotype.For example, and not by way of limitation, in the case of monocytes,such more normal phenotypes may include but are not limited to decreasedrates of LDL uptake, adhesion to endothelial cells, transmigration, foamcell formation,-fatty streak formation, and production by foam cells ofgrowth factors such as bFGF, IGF-I, VEGF, IL-1, M-CSF, TGFβ, TGFα, TNFα,HB-EGF, PDGF, IFN-γ, and GM-CSF. Transmigration rates, for example, maybe measured using the in vitro system of Navab et al., described inSection 5.1.1.3, above, by quantifying the number of monocytes thatmigrate across the endothelial monolayer and into the collagen layer ofthe subendothelial space.

[0248] In addition, animal-based cardiovascular disease systems, such asthose described, above, in Section 5.4.4.1, may be used to identifycompounds capable of ameliorating cardiovascular disease symptoms. Suchanimal models may be used as test substrates for the identification ofdrugs, pharmaceuticals, therapies, and interventions which may beeffective in treating cardiovascular disease. For example, animal modelsmay be exposed to a compound, suspected of exhibiting an ability toameliorate cardiovascular disease symptoms, at a sufficientconcentration and for a time sufficient to elicit such an ameliorationof cardiovascular disease symptoms in the exposed animals. The responseof the animals to the exposure may be monitored by assessing thereversal of disorders associated with cardiovascular disease, forexample, by counting the number of atherosclerotic plaques and/ormeasuring their size before and after treatment.

[0249] With regard to intervention, any treatments which reverse anyaspect of cardiovascular disease symptoms should be considered ascandidates for human cardiovascular disease therapeutic intervention.Dosages of test agents may be determined by deriving dose-responsecurves, as discussed in Section 5.7.1, below.

[0250] Additionally, gene expression patterns may be utilized to assessthe ability of a compound to ameliorate cardiovascular disease symptoms.For example, the expression pattern of one or more fingerprint genes mayform part of a “fingerprint profile” which may be then be used in suchan assessment. “Fingerprint profile”, as used herein, refers to thepattern of mRNA expression obtained for a given tissue or cell typeunder a given set of conditions. Such conditions may include, but arenot limited to, atherosclerosis, ischemia/reperfusion, hypertension,restenosis, and arterial inflammation, including any of the control orexperimental conditions described in the paradigms of Section5.1.1,above. Fingerprint profiles may be generated, for example, byutilizing a differential display procedure, as discussed, above, inSection 5.1.2, Northern analysis and/or RT-PCR. Any of the genesequences described, above, in Section 5.4.1. may be used as probesand/or PCR primers for the generation and corroboration of suchfingerprint profiles.

[0251] Fingerprint profiles may be characterized for known states,either cardiovascular disease or normal, within the cell- and/oranimal-based model systems. Subsequently, these known fingerprintprofiles may be compared to ascertain the effect a test compound has tomodify such fingerprint profiles, and to cause the profile to moreclosely resemble that of a more desirable fingerprint.

[0252] For example, administration of a compound may cause thefingerprint profile of a cardiovascular disease model system to moreclosely resemble the control system. Administration of a compound may,alternatively, cause the fingerprint profile of a control system tobegin to mimic a cardiovascular disease state. Such a compound may, forexample, be used in further characterizing the compound of interest, ormay be used in the generation of additional animal models.

[0253] 5.5.5. Monitoring of Effects During Clinical Trials

[0254] Monitoring the influence of compounds on cardiovascular diseasestates may be applied not only in basic drug screening, but also inclinical trials. In such clinical trials, the expression of a panel ofgenes that have been discovered in one of the paradigms described inSection 5.1.1.1 through 5.1.1.6 may be used as a “read out” of aparticular drug's effect on a cardiovascular disease state.

[0255] For example, and not by way of limitation, Paradigm A providesfor the identification of fingerprint genes that are up-regulated inmonocytes treated with oxidized LDL. Thus, to study the effect ofanti-oxidant drugs, for example, in a clinical trial, blood may be drawnfrom patients before and at different stages during treatment with sucha drug. Their monocytes may then be isolated and RNA prepared andanalyzed by differential display as described in Sections 6.1.1 and6.1.2. The levels of expression of these fingerprint genes may bequantified by Northern blot analysis or RT-PCR, as described in Section6.1.2, or by one of the methods described in Section 5.8.1, oralternatively by measuring the amount of protein produced, by one of themethods described in Section 5.8.2. In this way, the fingerprintprofiles may serve as surrogate markers indicative of the physiologicalresponse of monocytes that have taken up oxidized LDL. Accordingly, thisresponse state may be determined before, and at various points during,drug treatment. This method is described in further detail in theexample in Section 10, below.

[0256] This method may also be applied to the other paradigms disclosedherein. For example, and not by way of limitation, the fingerprintprofile of Paradigm B reveals that bcl-2 and glutathione peroxidase areboth down-regulated in the monocytes of patients exposed to a high lipiddiet, e.g. cholesterol or fat, that leads to high serum LDL levels.Drugs may be tested, for example, for their ability to ameliorate theeffects of hypercholesterolemia in clinical trials. Patients with highLDL levels may have their monocytes isolated before, and at differentstages after, drug treatment. The drug's efficacy may be measured bydetermining the degree of restored expression of bcl-2 and glutathioneperoxidase, as described above for the Paradigm A fingerprint profile.

[0257] 5.6. Compounds and Methods for Treatment of CardiovascularDisease

[0258] Described below are methods and compositions wherebycardiovascular disease symptoms may be ameliorated. Certaincardiovascular diseases are brought about, at least in part, by anexcessive level of gene product, or by the presence of a gene productexhibiting an abnormal or excessive activity. As such, the reduction inthe level and/or activity of such gene products would bring about theamelioration of cardiovascular disease symptoms. Techniques for thereduction of target gene expression levels or target gene productactivity levels are discussed in Section 5.6.1, below.

[0259] Alternatively, certain other cardiovascular diseases are broughtabout, at least in part, by the absence or reduction of the level ofgene expression, or a reduction in the level of a gene product'sactivity. As such, an increase in the level of gene expression and/orthe activity of such gene products would bring about the amelioration ofcardiovascular disease symptoms. Techniques for increasing target geneexpression levels or target gene product activity levels are discussedin Section 5.6.2, below.

[0260] 5.6.1. Compounds that Inhibit Expression, Synthesis or Activityof Mutant Target Gene Activity

[0261] As discussed above, target genes involved in cardiovasculardisease disorders can cause such disorders via an increased level oftarget gene activity. As summarized in Table 1, above, and detailed inthe examples in Sections 8 and 9, below, a number of genes are now knownto be up-regulated in endothelial cells under disease conditions.Specifically, rchd005, rchd024, rchd032, and rchd036 are allup-regulated in endothelial cells treated with IL-1.Furthermore,rchd502, rchd523, rchd528, rchd534, and endoperoxide synthase are allup-regulated in endothelial cells subjected to shear stress. A varietyof techniques may be utilized to inhibit the expression, synthesis, oractivity of such target genes and/or proteins.

[0262] For example, compounds such as those identified through assaysdescribed, above, in Section 5.5, which exhibit inhibitory activity, maybe used in accordance with the invention to ameliorate cardiovasculardisease symptoms. As discussed in Section 5.5, above, such molecules mayinclude, but are not limited to small organic molecules, peptides,antibodies, and the like. Inhibitory antibody techniques are described,below, in Section 5.6.1.2.

[0263] For example, compounds can be administered that compete withendogenous ligand for the rchd523 gen-product. The resulting reductionin the amount of ligand-bound rchd523 gene transmembrane protein willmodulated endothelial cell physiology. Compounds that can beparticularly useful for this purpose include, for example, solubleproteins or peptides, such as peptides comprising one or more of theextracellular domains, or portions and/or analogs thereof, of therchd523 gene product, including, for example, soluble fusion proteinssuch as Ig-tailed fusion proteins. (For a discussion of the productionof Ig-tailed fusion proteins, see, for example, U.S. Pat. No.5,116,964.). Alternatively, compounds, such as ligand analogs orantibodies, that bind to the rchd523 gene product receptor site, but donot activate the protein, (e.g., receptor-ligand antagonists) can beeffective in inhibiting rchd523 gene product activity.

[0264] Further, antisense and ribozyme molecules which inhibitexpression of the target gene may also be used in accordance with theinvention to inhibit the aberrant target gene activity. Such techniquesare described, below, in Section 5.6.1.1. Still further, also asdescribed, below, in Section 5.6.1.1, triple helix molecules may beutilized in inhibiting the aberrant target gene activity.

[0265] 5.6.1.1. Inhibitory Antisense, Ribozyme and Triple HelixApproaches

[0266] Among the compounds which may exhibit the ability to amelioratecardiovascular disease symptoms are antisense, ribozyme, and triplehelix molecules. Such molecules may be designed to reduce or inhibitmutant target gene activity. Techniques for the production and use ofsuch molecules are well known to those of skill in the art.

[0267] Anti-sense RNA and DNA molecules act to directly block thetranslation of mRNA by hybridizing to targeted mRNA and preventingprotein translation. With respect to antisense DNA,oligodeoxyribonucleotides derived from the translation initiation site,e.g., between the −10 and +10 regions of the target gene nucleotidesequence of interest, are preferred.

[0268] Ribozymes are enzymatic RNA molecules capable of catalyzing thespecific cleavage of RNA. The mechanism of ribozyme action involvessequence specific hybridization of the ribozyme molecule tocomplementary target RNA, followed by an endonucleolytic cleavage. Thecomposition of ribozyme molecules must include one or more sequencescomplementary to the target gene mRNA, and must include the well knowncatalytic sequence responsible for mRNA cleavage. For this sequence, seeU.S. Pat. No. 5,093,246, which is incorporated by reference herein inits entirety. As such within the scope of the invention are engineeredhammerhead motif ribozyme molecules that specifically and efficientlycatalyze endonucleolytic cleavage of RNA sequences encoding target geneproteins.

[0269] Specific ribozyme cleavage sites within any potential RNA targetare initially identified by scanning the molecule of interest forribozyme cleavage sites which include the following sequences, GUA, GUUand GUC. Once identified, short RNA sequences of between 15 and20ribonucleotides corresponding to the region of the target genecontaining the cleavage site may be evaluated for predicted structuralfeatures, such as secondary structure, that may render theoligonucleotide sequence unsuitable. The suitability of candidatesequences may also be evaluated by testing their accessibility tohybridization with complementary oligonucleotides, using ribonucleaseprotection assays.

[0270] Nucleic acid molecules to be used in triple helix formation forthe inhibition of transcription should be single stranded and composedof deoxyribonucleotides. The base composition of these oligonucleotidesmust be designed to promote triple helix formation via Hoogsteen basepairing rules, which generally require sizeable stretches of eitherpurines or pyrimidines to be present on one strand of a duplex.Nucleotide sequences may be pyrimidine-based, which will result in TATand CGC⁺ triplets across the three associated strands of the resultingtriple helix. The pyrimidine-rich molecules provide base complementarityto a purine-rich region of a single strand of the duplex in a parallelorientation to that strand. In addition, nucleic acid molecules may bechosen that are purine-rich, for example, containing a stretch of Gresidues. These molecules will form a triple helix with a DNA duplexthat is rich in GC paris, in which the majority of the purine residuesare located on a single strand of the targeted duplex, resulting in GGCtriplets across the three strands in the triplex.

[0271] Alternatively, the potential sequences that can be targeted fortriple helix formation may be increased by creating a so called“switchback” nucleic acid molecule. Switchback molecules are synthesizedin an alternating 5′-3′, 3′-5′ manner, such that they base pair withfirst one strand of a duplex and then the other, eliminating thenecessity for a sizeable stretch of either purines or pyrimidines to bepresent on one strand of a duplex.

[0272] It is possible that the antisense, ribozyme, and/or triple helixmolecules described herein may reduce or inhibit the transcription(triple helix) and/or translation (antisense, ribozyme) of mRNA producedby both normal and mutant target gene alleles. In order to ensure thatsubstantially normal levels of target gene activity are maintained,nucleic acid molecules that encode and express target gene polypeptidesexhibiting normal activity may be introduced into cells via gene therapymethods such as those described, below, in Section 5.7. that do notcontain sequences susceptible to whatever antisense, ribozyme, or triplehelix treatments are being utilized. Alternatively, it may be preferableto coadminister normal target gene protein into the cell or tissue inorder to maintain the requisite level of cellular or tissue target geneactivity.

[0273] Anti-sense RNA and DNA, ribozyme, and triple helix molecules ofthe invention may be prepared by any method known in the art for thesynthesis of DNA and RNA molecules. These include techniques forchemically synthesizing oligodeoxyribonucleotides andoligoribonucleotides well known in the art such as for example solidphase phosphoramidite chemical synthesis. Alternatively, RNA moleculesmay be generated by in vitro and in vivo transcription of DNA sequencesencoding the antisense RNA molecule. Such DNA sequences may beincorporated into a wide variety of vectors which incorporate suitableRNA polymerase promoters such as the T7 or SP6 polymerase promoters.Alternatively, antisense cDNA constructs that synthesize antisense RNAconstitutively or inducibly, depending on the promoter used, can beintroduced stably into cell lines.

[0274] Various well-known modifications to the DNA molecules may beintroduced as a means of increasing intracellular stability andhalf-life. Possible modifications include but are not limited to theaddition of flanking sequences of ribonucleotides ordeoxyribonucleotides to the 5′ and/or 3′ ends of the molecule or the useof phosphorothioate or 2′ O-methyl rather than phosphodiesteraselinkages within the oligodeoxyribonucleotide backbone.

[0275] 5.6.1.2. Antibodies for Target Gene Products

[0276] Antibodies that are both specific for target gene protein andinterfere with its activity may be used to inhibit target gene function.Such antibodies may be generated using standard techniques described inSection 5.4.3., supra, against the proteins themselves or againstpeptides corresponding to portions of the proteins. Such antibodiesinclude but are not limited to polyclonal, monoclonal, Fab fragments,single chain antibodies, chimeric antibodies, etc.

[0277] In instances where the target gene protein is intracellular andwhole antibodies are used, internalizing antibodies may be preferred.However, lipofectin liposomes may be used to deliver the antibody or afragment of the Fab region which binds to the target gene epitope intocells. Where fragments of the antibody are used, the smallest inhibitoryfragment which binds to the target protein's binding domain ispreferred. For example, peptides having an amino acid sequencecorresponding to the domain of the variable region of the antibody thatbinds to the target gene protein may be used. Such peptides may besynthesized chemically or produced via recombinant DNA technology usingmethods well known in the art (e.g., see Creighton, 1983, supra; andSambrook et al., 1989, supra). Alternatively, single chain neutralizingantibodies which bind to intracellular target gene epitopes may also beadministered. Such single chain antibodies may be administered, forexample, by expressing nucleotide sequences encoding single-chainantibodies within the target cell population by utilizing, for example,techniques such as those described in Marasco et al. (Marasco, W. etal., 1993, Proc. Natl. Acad. Sci. USA 90:7889-7893).

[0278] In some instances, the target gene protein is extracellular, oris a transmembrane protein, such as the rchd523 gene product. Antibodiesthat are specific for one or more extracellular domains of the rchd523gene product, for example, and that interfere with its activity, areparticularly useful in treating cardiovascular disease. Such antibodiesare especially efficient because they can access the target domainsdirectly from the bloodstream. Any of the administration techniquesdescribed, below in Section 5.7which are appropriate for peptideadministration may be utilized to effectively administer inhibitorytarget gene antibodies to their site of action.

[0279] 5.6.2. Methods for Restoring Target Gene Activity

[0280] Target genes that cause cardiovascular disease may beunderexpressed within cardiovascular disease situations. As summarizedin Table 1, above, and detailed in the example in Sections 7, below,several genes are now known to be down-regulated in monocytes underdisease conditions. Specifically, bcl-2 and glutathione peroxidase geneexpression is down-regulated in the monocytes of patients exposed to ahigh lipid diet, e.g. cholesterol or fat, that leads to high serum LDLlevels. Alternatively, the activity of target gene products may bediminished, leading to the development of cardiovascular diseasesymptoms. Described in this Section are methods whereby the level oftarget gene activity may be increased to levels wherein cardiovasculardisease symptoms are ameliorated. The level of gene activity may beincreased, for example, by either increasing the level of target geneproduct present or by increasing the level of active target gene productwhich is present.

[0281] For example, a target gene protein, at a level sufficient toameliorate cardiovascular disease symptoms may be administered to apatient exhibiting such symptoms. Any of the techniques discussed,below, in Section 5.7, may be utilized for such administration. One ofskill in the art will readily know how to determine the concentration ofeffective, non-toxic doses of the normal target gene protein, utilizingtechniques such as those described, below, in Section 5.7.1.

[0282] Additionally, RNA sequences encoding target gene rotein may bedirectly administered to a patient exhibiting ardiovascular diseasesymptoms, at a concentration sufficient to produce a level of targetgene protein such that cardiovascular disease symptoms are ameliorated.Any of he techniques discussed, below, in Section 5.7, which achieveintracellular administration of compounds, such as, for example,liposome administration, may be utilized for the administration of suchRNA molecules. The RNA molecules may be produced, for example, byrecombinant techniques such as those described, above, in Section 5.4.2.

[0283] Further, patients may be treated by gene replacement therapy. Oneor more copies of a normal target gene, or a portion of the gene thatdirects the production of a normal target gene protein with target genefunction, may be inserted into cells using vectors which include, butare not limited to adenovirus, adeno-associated virus, and retrovirusvectors, in addition to other particles that introduce DNA into cells,such as liposomes. Additionally, techniques such as those describedabove may be utilized for the introduction of normal target genesequences into human cells.

[0284] Cells, preferably, autologous cells, containing normal targetgene expressing gene sequences may then be introduced or reintroducedinto the patient at positions which allow for the amelioration ofcardiovascular disease symptoms. Such cell replacement techniques may bepreferred, for example, when the target gene product is a secreted,extracellular gene product.

[0285] 5.7. Pharmaceutical Preparations and Methods of Administration

[0286] The identified compounds that inhibit target gene expression,synthesis and/or activity can be administered to a patient attherapeutically effective doses to treat or ameliorate cardiovasculardisease. A therapeutically effective dose refers to that amount of thecompound ufficient to result in amelioration of symptoms ofardiovascular disease.

[0287] 5.7.1. Effective Dose

[0288] Toxicity and therapeutic efficacy of such compounds an bedetermined by standard pharmaceutical procedures in ell cultures orexperimental animals, e.g., for determining he LD₅₀ (the dose lethal to50% of the population) and the ED₅₀ (the dose therapeutically effectivein 50% of the population). The dose ratio between toxic and therapeuticeffects is the therapeutic index and it can be expressed as the ratioLD₅₀/ED₅₀. Compounds which exhibit large-therapeutic indices arepreferred. While compounds that exhibit toxic side effects may be used,care should be taken to design a delivery system that targets suchcompounds to the site of affected tissue in order to minimize potentialdamage to uninfected cells and, thereby, reduce side effects.

[0289] The data obtained from the cell culture assays and animal studiescan be used in formulating a range of dosage for use in humans. Thedosage of such compounds lies preferably within a range of circulatingconcentrations that include the ED₅₀ with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized. For any compound usedin the method of the invention, the therapeutically effective dose canbe estimated initially from cell culture assays. A dose may beformulated in animal models to achieve a circulating plasmaconcentration range that includes the IC₅₀ (i.e., the concentration ofthe test compound which achieves a half-maximal inhibition of symptoms)as determined in cell culture. Such information can be used to moreaccurately determine useful doses in humans. Levels in plasma may bemeasured, for example, by high performance liquid chromatography.

[0290] 5.7.2. Formulations and Use

[0291] Pharmaceutical compositions for use in accordance with thepresent invention may be formulated in conventional manner using one ormore physiologically acceptable carriers or excipients.

[0292] Thus, the compounds and their physiologically acceptable saltsand solvates may be formulated for administration by inhalation orinsufflation (either through the mouth or the nose) or oral, buccal,parenteral or rectal administration.

[0293] For oral administration, the pharmaceutical compositions may takethe form of, for example, tablets or capsules prepared by conventionalmeans with pharmaceutically acceptable excipients such as binding agents(e.g., pregelatinised maize starch, polyvinylpyrrolidone orhydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystallinecellulose or calcium hydrogen phosphate); lubricants (e.g., magnesiumstearate, talc or silica); disintegrants (e.g., potato starch or sodiumstarch glycolate); or wetting agents (e.g. sodium lauryl sulphate). Thetablets may be coated by methods well known in the art. Liquidpreparations for oral administration may take the form of, for example,solutions, syrups or suspensions, or they may be presented as a dryproduct for constitution with water or other suitable vehicle beforeuse. Such liquid preparations may be prepared by conventional means withpharmaceutically acceptable additives such as suspending agents (e.g.,sorbitol syrup, cellulose derivatives or hydrogenated edible fats);emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles(e.g., almond oil, oily esters, ethyl alcohol or fractionated vegetableoils); and

[0294] preservatives (e.g., methyl or propyl-p-hydroxybenzoates orsorbic acid). The preparations may also contain buffer salts, flavoring,coloring and sweetening agents as appropriate.

[0295] Preparations for oral administration may be suitably formulatedto give controlled release of the active compound.

[0296] For buccal administration the compositions may take the form oftablets or lozenges formulated in conventional manner.

[0297] For administration by inhalation, the compounds for use accordingto the present invention are conveniently delivered in the form of anaerosol spray presentation from pressurized packs or a nebuliser, withthe use of a suitable propellant, e.g., dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orother suitable gas. In the case of a pressurized aerosol the dosage unitmay be determined by providing a valve to deliver a metered amount.Capsules and cartridges of e.g. gelatin for use in an inhaler orinsufflator may be formulated containing a powder mix of the compoundand a suitable powder base such as lactose or starch.

[0298] The compounds may be formulated for parenteral administration byinjection, e.g., by bolus injection or continuous infusion. Formulationsfor injection may be presented in unit dosage form, e.g., in ampoules orin multi-dose containers, with an added preservative. The compositionsmay take such forms as suspensions, solutions or emulsions in oily oraqueous vehicles, and may contain formulatory agents such as suspending,stabilizing and/or dispersing agents. Alternatively, the activeingredient may be in powder form for constitution with a suitablevehicle, e.g., sterile pyrogen-free water, before use.

[0299] The compounds may also be formulated in rectal compositions suchas suppositories or retention enemas, e.g., containing conventionalsuppository bases such as cocoa butter or other glycerides.

[0300] In addition to the formulations described previously, thecompounds may also be formulated as a depot preparation. Such longacting formulations may be administered by implantation (for examplesubcutaneously or intramuscularly) or by intramuscular injection. Thus,for example, the compounds may be formulated with suitable polymeric orhydrophobic materials (for example as an emulsion in an acceptable oil)or ion exchange resins, or as sparingly soluble derivatives, forexample, as a sparingly soluble salt.

[0301] The compositions may, if desired, be presented in a pack ordispenser device which may contain one or more unit dosage formscontaining the active ingredient. The pack may for example comprisemetal or plastic foil, such as a blister pack. The pack or dispenserdevice may be accompanied by instructions for administration.

[0302] 5.8. Diagnosis of Cardiovascular Disease Abnormalities

[0303] A variety of methods may be employed, utilizing reagents such asfingerprint gene nucleotide sequences described in Section 5.4.1, andantibodies directed against differentially expressed and pathway genepeptides, as described, above, in Sections 5.4.2. (peptides) and 5.4.3.(antibodies). Specifically, such reagents may be used, for example, forthe detection of the presence of target gene mutations, or the detectionof either over or under expression of target gene mRNA.

[0304] The methods described herein may be performed, for example, byutilizing pre-packaged diagnostic kits comprising at least one specificfingerprint gene nucleic acid or anti-fingerprint gene antibody reagentdescribed herein, which may be conveniently used, e.g., in clinicalsettings, to diagnose patients exhibiting cardiovascular diseasesymptoms or at risk for developing cardiovascular disease.

[0305] Any cell type or tissue, preferably monocytes, endothelial cells,or smooth muscle cells, in which the fingerprint gene is expressed maybe utilized in the diagnostics described below.

[0306] 5.8.1. Detection of Fingerprint Gene Nucleic Acids

[0307] DNA or RNA from the cell type or tissue to be analyzed may easilybe isolated using procedures which are well known to those in the art.Diagnostic procedures may also be performed “in situ” directly upontissue sections (fixed and/or frozen) of patient tissue obtained frombiopsies or resections, such that no nucleic acid purification isnecessary. Nucleic acid reagents such as those described in Section 5.1.may be used as probes and/or primers for such in situ procedures (see,for example, Nuovo, G. J., 1992, PCR in situ hybridization: protocolsand applications, Raven Press, NY).

[0308] Fingerprint gene nucleotide sequences, either RNA or DNA, may,for example, be used in hybridization or amplification assays ofbiological samples to detect cardiovascular disease-related genestructures and expression. Such assays may include, but are not limitedto, Southern or Northern analyses, single stranded conformationalpolymorphism analyses, in situ hybridization assays, and polymerasechain reaction analyses. Such analyses may reveal both quantitativeaspects of the expression pattern of the fingerprint gene, andqualitative aspects of the fingerprint gene expression and/or genecomposition. That is, such aspects may include, for example, pointmutations, insertions, deletions, chromosomal rearrangements, and/oractivation or inactivation of gene expression.

[0309] Preferred diagnostic methods for the detection of fingerprintgene-specific nucleic acid molecules may involve for example, contactingand incubating nucleic acids, derived from the cell type or tissue beinganalyzed, with one or more labeled nucleic acid reagents as aredescribed in Section 5.1, under conditions favorable for the specificannealing of these reagents to their complementary sequences within thenucleic acid molecule of interest. Preferably, the lengths of thesenucleic acid reagents are at least 9 to 30 nucleotides. Afterincubation, all non-annealed nucleic acids are removed from the nucleicacid:fingerprint molecule hybrid. The presence of nucleic acids from thefingerprint tissue which have hybridized, if any such molecules exist,is then detected. Using such a detection scheme, the nucleic acid fromthe tissue or cell type of interest may be immobilized, for example, toa solid support such as a membrane, or a plastic surface such as that ona microtitre plate or polystyrene beads. In this case, after incubation,non-annealed, labeled fingerprint nucleic acid reagents of the typedescribed in Section 5.1. are easily removed. Detection of theremaining, annealed, labeled nucleic acid reagents is accomplished usingstandard technique well-known to those in the art.

[0310] Alternative diagnostic methods for the detection of fingerprintgene specific nucleic acid molecules may involve their amplification,e.g., by PCR (the experimental embodiment set forth in Mullis, K. B.,1987, U.S. Pat. No. 4,683,202), ligase chain reaction (Barany, F., 1991,Proc. Natl. Acad. Sci. USA 88:189-193), self sustained sequencereplication (Guatelli, J.C. et al., 1990, Proc. Natl. Acad. Sci. USA87:1874-1878), transcriptional amplification system (Kwoh, D. Y et al.,1989, Proc. Natl. Acad. Sci. USA 86:1173-1177), Q-Beta Replicase(Lizardi, P. M. et al., 1988, Bio/Technology 6:1197), or any othernucleic acid amplification method, followed by the detection of theamplified molecules using techniques well known to those of skill in theart. These detection schemes are especially useful for the detection ofnucleic acid molecules if such molecules are present in very lownumbers.

[0311] In one embodiment of such a detection scheme, a cDNA molecule isobtained from an RNA molecule of interest (e.g., by reversetranscription of the RNA molecule into cDNA). Cell types or tissues fromwhich such RNA may be isolated include any tissue in which wild typefingerprint gene is known to be expressed, including, but not limited,to monocytes, endothelium, and/or smooth muscle. A fingerprint sequencewithin the cDNA is then used as the template for a nucleic acidamplification reaction, such as a PCR amplification reaction, or thelike. The nucleic acid reagents used as synthesis initiation reagents(e.g., primers) in the reverse transcription and nucleic acidamplification steps of this method are chosen from among the fingerprintgene nucleic acid reagents described in Section 5.1. The preferredlengths of such nucleic acid reagents are at least 15-30 nucleotides.For detection of the amplified product, the nucleic acid amplificationmay be performed using radioactively or non-radioactively labelednucleotides. Alternatively, enough amplified product may be made suchthat the product may be visualized by standard ethidium bromide stainingor by utilizing any other suitable nucleic acid staining method.

[0312] In addition to methods which focus primarily on the detection ofone nucleic acid sequence, fingerprint profiles, as discussed in Section5.5.4, may also be assessed in such detection schemes. Fingerprintprofiles may be generated, for example, by utilizing a differentialdisplay procedure, as discussed, above, in Section 5.1.2, Northernanalysis and/or RT-PCR. Any of the gene sequences described, above, inSection 5.4.1. may be used as probes and/or PCR primers for thegeneration and corroboration of such fingerprint profiles.

[0313] 5.8.2. Detection of Fingerprint Gene Peptides

[0314] Antibodies directed against wild type or mutant fingerprint genepeptides, which are discussed, above, in Section 5.4.3, may also be usedas cardiovascular disease diagnostics and prognostics, as described, forexample, herein. Such diagnostic methods, may be used to detectabnormalities in the level of fingerprint gene protein expression, orabnormalities in the structure and/or tissue, cellular, or subcellularlocation of fingerprint gene protein. Structural differences mayinclude, for example, differences in the size, electronegativity, orantigenicity of the mutant fingerprint gene protein relative to thenormal fingerprint gene protein.

[0315] Protein from the tissue or cell type to be analyzed may easily beisolated using techniques which are well known to those of skill in theart. The protein isolation methods employed herein may, for example, besuch as those described in Harlow and Lane (Harlow, E. and Lane, D.,1988, “Antibodies: A Laboratory Manual”, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y.), which is incorporated herein byreference in its entirety.

[0316] Preferred diagnostic methods for the detection of wild type ormutant fingerprint gene peptide molecules may involve, for example,immunoassays wherein fingerprint gene peptides are detected by theirinteraction with an anti-fingerprint gene specific peptide antibody.

[0317] For example, antibodies, or fragments of antibodies, such asthose described, above, in Section 5.4.3, useful in the presentinvention may be used to quantitatively or qualitatively detect thepresence of wild type or mutant fingerprint gene peptides. This can beaccomplished, for example, by immunofluorescence techniques employing afluorescently labeled antibody (see below) coupled with lightmicroscopic, flow cytometric, or fluorimetric detection. Such techniquesare especially preferred if the fingerprint gene peptides are expressedon the cell surface.

[0318] The antibodies (or fragments thereof) useful in the presentinvention may, additionally, be employed histologically, as inimmunofluorescence or immunoelectron microscopy, for in situ detectionof fingerprint gene peptides. In situ detection may be accomplished byremoving a histological specimen from a patient, and applying thereto alabeled antibody of the present invention. The antibody (or fragment) ispreferably applied by overlaying the labeled antibody (or fragment) ontoa biological sample. Through the use of such a procedure, it is possibleto determine not only the presence of the fingerprint gene peptides, butalso their distribution in the examined tissue. Using the presentinvention, those of ordinary skill will readily perceive that any of awide variety of histological methods (such as staining procedures) canbe modified in order to achieve such in situ detection.

[0319] Immunoassays for wild type or mutant fingerprint gene peptidestypically comprise incubating a biological sample, such as a biologicalfluid, a tissue extract, freshly harvested cells, or cells which havebeen incubated in tissue culture, in the presence of a detectablylabeled antibody capable of identifying fingerprint gene peptides, anddetecting the bound antibody by any of a number of techniques well knownin the art.

[0320] The biological sample may be brought in contact with andimmobilized onto a solid phase support or carrier such asnitrocellulose, or other solid support which is capable of immobilizingcells, cell particles or soluble proteins. The support may then bewashed with suitable buffers followed by treatment with the detectablylabeled fingerprint gene specific antibody. The solid phase support maythen be washed with the buffer a second time to remove unbound antibody.The amount of bound label on solid support may then be detected byconventional means.

[0321] By “solid phase support or carrier” is intended any supportcapable of binding an antigen or an antibody. Well-known supports orcarriers include glass, polystyrene, polypropylene, polyethylene,dextran, nylon, amylases, natural and modified celluloses,polyacrylamides, gabbros, and magnetite. The nature of the carrier canbe either soluble to some extent or insoluble for the purposes of thepresent invention. The support material may have virtually any possiblestructural configuration so long as the coupled molecule is capable ofbinding to an antigen or antibody. Thus, the support configuration maybe spherical, as in a bead, or cylindrical, as in the inside surface ofa test tube, or the external surface of a rod. Alternatively, thesurface may be flat such as a sheet, test strip, etc. Preferred supportsinclude polystyrene beads. Those skilled in the art will know many othersuitable carriers for binding antibody or antigen, or will be able toascertain the same by use of routine experimentation.

[0322] The binding activity of a given lot of anti-wild type or mutantfingerprint gene peptide antibody may be determined according to wellknown methods. Those skilled in the art will be able to determineoperative and optimal assay conditions for each determination byemploying routine experimentation.

[0323] One of the ways in which the fingerprint gene peptide-specificantibody can be detectably labeled is by linking the same to an enzymeand use in an enzyme immunoassay (EIA) (Voller, “The Enzyme LinkedImmunosorbent Assay (ELISA)”, Diagnostic Horizons 2:1-7,1978,Microbiological Associates Quarterly Publication, Walkersville,Md.; Voller, et al., J. Clin. Pathol. 31:507-520 (1978); Butler, Meth.Enzymol. 73:482-523 (1981); Maggio, (ed.) Enzyme Immunoassay, CRC Press,Boca Raton, Fla., 1980; Ishikawa, et al., (eds.) Enzyme Immunoassay,Kgaku Shoin, Tokyo, 1981). The enzyme which is bound to the antibodywill react with an appropriate substrate, preferably a chromogenicsubstrate, in such a manner as to produce a chemical moiety which can bedetected, for example, by spectrophotometric, fluorimetric or by visualmeans. Enzymes which can be used to detectably label the antibodyinclude, but are not limited to, malate dehydrogenase, staphylococcalnuclease, delta-5-steroid isomerase, yeast alcohol dehydrogenase,alpha-glycerophosphate, dehydrogenase, triose phosphate isomerase,horseradish peroxidase, alkaline phosphatase, asparaginase, glucoseoxidase, beta-galactosidase, ribonuclease, urease, catalase,glucose-6-phosphate dehydrogenase, glucoamylase andacetylcholinesterase. The detection can be accomplished by colorimetricmethods which employ a chromogenic substrate for the enzyme. Detectionmay also be accomplished by visual comparison of the extent of enzymaticreaction of a substrate in comparison with similarly prepared standards.

[0324] Detection may also be accomplished using any of a variety ofother immunoassays. For example, by radioactively labeling theantibodies or antibody fragments, it is possible to detect fingerprintgene wild type or mutant peptides through the use of a radioimmunoassay(RIA) (see, for example, Weintraub, B., Principles of Radioimmunoassays,Seventh Training Course on Radioligand Assay Techniques, The EndocrineSociety, March, 1986, which is incorporated by reference herein). Theradioactive isotope can be detected by such means as the use of a gammacounter or a scintillation counter or by autoradiography.

[0325] It is also possible to label the antibody with a fluorescentcompound. When the fluorescently labeled antibody is exposed to light ofthe proper wave length, its presence can then be detected due tofluorescence. Among the most commonly used fluorescent labelingcompounds are fluorescein isothiocyanate, rhodamine, phycoerythrin,phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine.

[0326] The antibody can also be detectably labeled using fluorescenceemitting metals such as ¹⁵²Eu, or others of the lanthanide series. Thesemetals can be attached to the antibody using such metal chelating groupsas diethylenetriaminepentacetic acid (DTPA) orethylenediaminetetraacetic acid (EDTA).

[0327] The antibody also can be detectably labeled by coupling it to achemiluminescent compound. The presence of the chemiluminescent-taggedantibody is then determined by detecting the presence of luminescencethat arises during the course of a chemical reaction. Examples ofparticularly useful chemiluminescent labeling compounds are luminol,isoluminol, theromatic acridinium ester, imidazole, acridinium salt andoxalate ester.

[0328] Likewise, a bioluminescent compound may be used to label theantibody of the present invention. Bioluminescence is a type ofchemiluminescence found in biological systems in, which a catalyticprotein increases the efficiency of the chemiluminescent reaction. Thepresence of a bioluminescent protein is determined by detecting thepresence of luminescence. Important bioluminescent compounds forpurposes of labeling are luciferin, luciferase and aequorin.

[0329] 5.8.3. Imaging Cardiovascular Disease Conditions

[0330] In some cases, differentially expressed gene products identifiedherein may be up-regulated under cardiovascular disease conditions andexpressed on the surface of the affected tissue. Such target geneproducts allow for the non-invasive imaging of damaged or diseasedcardiovascular tissue for the purposed of diagnosis and directing oftreatment of the disease. For example, such differentially expressedgene products may include but are not limited to atherosclerosisspecific adhesion molecules responsible for atherogenesis, or monocytescavenger receptors that are up-regulated in response to oxidized LDL,which are discussed in Section 2, above. Alternatively, other suchsurface proteins may be specifically up-regulated in tissues sufferingfrom ischemia/reperfusion or other tissues with atherosclerotic orrestenotic lesions.

[0331] As described in the example in Section 9, below, the rchd523 geneis a gene that is up-regulated in endothelial cells under shear stress.Furthermore, the rchd523 gene encodes a novel G protein-coupledreceptor, containing an extracellular amino terminal domain, in additionto multiple transmembrane domains. The rchd523 gene product, therefore,provides an excellent tool for imaging cardiovascular diseaseconditions. An example illustrating the use of this method in accordancewith the invention is provided in Section 11, below.

[0332] Monoclonal antibodies, as described in Section 5.6.1.2, above,which specifically bind to such surface proteins, such as the rchd523gene product, may be used for the diagnosis of cardiovascular disease byin vivo tissue imaging techniques. An antibody specific for a targetgene product, or preferably an antigen binding fragment thereof, isconjugated to a label (e.g., a gamma emitting radioisotope) whichgenerates a detectable signal and administered to a subject (human oranimal) suspected of having cardiovascular disease. After sufficienttime to allow the detectably-labeled antibody to localize at thediseased or damaged tissue site (or sites), the signal generated by thelabel is detected by a photoscanning device. The detected signal is thenconverted to an image of the tissue. This image makes it possible tolocalize the tissue in vivo. This data can then be used to develop anappropriate therapeutic strategy.

[0333] Antibody fragments, rather than whole antibody molecules, aregenerally preferred for use in tissue imaging. Antibody fragmentsaccumulate at the tissue(s) more rapidly because they are distributedmore readily than are entire antibody molecules. Thus an image can beobtained in less time than is possible using whole antibody. Thesefragments are also cleared more rapidly from tissues, resulting in alower background signal. See, e.g., Haber et al., U.S. Pat. No.4,036,945; Goldenberg et al., U.S. Pat. No. 4,331,647. The divalentantigen binding fragment (Fab′)₂ and the monovalent Fab are especiallypreferred. Such fragments can be prepared by digestion of the wholeimmunoglobulin molecule with the enzymes pepsin or papain according toany of several well known protocols. The types of labels that aresuitable for conjugation to a monoclonal antibody for diseased ordamaged tissue localization include, but are not limited to radiolabels(i.e., radioisotopes), fluorescent labels and biotin labels.

[0334] Among the radioisotopes that can be used to label antibodies orantibody fragments, gamma-emitters, positron-emitters, X-ray-emittersand fluorescence-emitters are suitable for localization. Suitableradioisotopes for labeling antibodies include Iodine-131, Iodine-123,Iodine-125, Iodine-126, Iodine-133, Bromine-77, Indium-111, Indium-113m,Gallium-67, Gallium-68, Ruthenium-95, Ruthenium-97, Ruthenium-103,Ruthenium-105, Mercury-107, Mercury-203, Rhenium-99m, Rhenium-105,Rhenium-101, Tellurium-121m, Tellurium-122m, Tellurium-125m,Thulium-165, Thulium-167, Thulium-168, Technetium-99m and Fluorine-18.The halogens can be used more or less interchangeably as labels sincehalogen-labeled antibodies and/or normal immunoglobulins would havesubstantially the same kinetics and distribution and similar metabolism.

[0335] The gamma-emitters Indium-111 and Technetium-99m are preferredbecause these radiometals are detectable with a gamma camera and havefavorable half lives for imaging in vivo. Antibody can be labelled withIndium-111 or Technetium-99m via a conjugated metal chelator, such asDTPA (diethlenetriaminepentaacetic acid). See Krejcarek et al., 1977,Biochem. Biophys. Res. Comm. 77:581; Khaw et al., 1980,Science 209:295;Gansow et al., U.S. Pat. No. 4,472,509; Hnatowich, U.S. Pat. No.4,479,930, the teachings of which are incorporated herein by reference.

[0336] Fluorescent compounds that are suitable for conjugation to amonoclonal antibody include fluorescein sodium, fluoresceinisothiocyanate, and Texas Red sulfonyl chloride. See, DeBelder & Wik,1975, Carbohydrate Research 44:254-257. Those skilled in the art willknow, or will be able to ascertain with no more than routineexperimentation, other fluorescent compounds that are suitable forlabeling monoclonal antibodies.

6. EXAMPLE: IDENTIFICATION OF GENES DIFFERENTIALLY EXPRESSED IN RESPONSETO PARADIGM A: IN VITRO FOAM CELL PARADIGM

[0337] According to the invention, differential display may be used todetect genes that are differentially expressed in monocytes that weretreated so as to simulate the conditions under which foam cells developduring atherogenesis.

[0338] 6.1. Materials and Methods

[0339] 6.1.1. Cell Isolation and Culturing

[0340] Blood (˜200 ml) was drawn into chilled 20 ml vacutainer tubes towhich 3 ml of citrate phosphate dextrose (Sigma) was added. Blood wasthen pooled into 50 ml tubes and spun in the Beckman GS-6R at 1250 RPMfor 15 minutes at 4° C. The upper clear layer (˜25 ml) was then removedwith a pipette and discarded and replaced with the same volume of 4° C.PBS. The blood was then mixed, and spun again at 2680RPM for 15 minutesat 4° C. The upper layer was then removed and discarded, and the buffycoat at the interface was removed in ˜5 ml and placed in a separate 50ml tube, and the pipette was washed with 20 ml PBS. Cells were added toa T flask and stored at 4° C. for 16 hours. A small aliquot of the cellswere then removed and counted using a hemacytometer. The final red bloodcell concentration in the buffy coat population was then adjusted to1.5×10⁹/ml with PBS, the cells were added to Leucoprep tubes (BectonDickinson) after being allowed to come to room temperature, and spun at2300RPM for 25 minutes at 25° C. The upper clear layer was removed anddiscarded and the turbid layer over the gel was removed and pooled in 50ml tubes. Samples were then diluted 1 to 50 ml with PBS (25° C.) andspun at 1000 RPM for 10 minutes. The supernatant was then removed, andthe pellet was resuspended in 50 ml PBS. This procedure was repeated 3more times. After the last spin, the cells were resuspended in a smallvolume of PBS and counted.

[0341] Tissue culture dishes were coated with bovine collagen beforemonocytes were plated out. ⅙ volume of 7× RPMI (JRH Biosciences) wasadded to Vitrogen 100 collagen (Celtrix) which was then diluted 1:10with RPMI to a final concentration of 0.35 mg/ml. Collagen mixture wasthen added to plates (2.5 ml/100 mm dish) and placed at 37° C. for atleast one hour to allow for gel formation. After gel formation has takenplace, the RPMI was removed and cells were added in RPMI/10% plasmaderived serum (PDS). PDS was prepared by drawing blood into chilledevacuated tubes containing {fraction (1/10)}th volume 3.8% sodiumcitrate. Blood was then transferred into new Sorvall tubes and spun at14,000-16,000 RPM for 20 minutes at 4° C. Plasma layer was removed andpooled in new tubes to which {fraction (1/50)}th volume 1M CaCl₂ wasadded. Plasma was mixed and aliquoted into new Sorvall tubes andincubated at 37% for 2 hours to allow for fibrin clot formation. Theclot was then disturbed with a pipette to allow it to contract and tubeswere spun at 14,500 RPM for 20 minutes at 25° C. Supernatant wascollected, pooled, and heat inactivated at 56° C. prior to sterilefiltration and freezing.

[0342] Purified human monocytes were cultured in 10% PDS/RPMI containing5 units/ml of Genzyme recombinant human MCSF for 5 days before beingtreated with LDL, oxidized LDL, acetylated LDL (all LDL at 50 μg/ml),lysophosphatidylcholine (Sigma, 37.5 μM), or homocysteine (Sigma, 1 mM).After incubation with these reagents for periods ranging from 2 hours upto 3 days, the media was withdrawn and the cells were dissolved in RNAlysis buffer and RNA was prepared as described, above, in Section 6.1.

[0343] Lipoproteins

[0344] For oxidation, human LDL (Sigma) was first diluted to 1 mg/mlwith PBS and then dialyzed against PBS at 4° C. overnight. LDL was thendiluted to 0.3 mg/ml with PBS. CuSO₄.5H₂O was then added to 5 μM finalconcentration, and the solution was incubated in a T flask in a 37° C.incubator for 24 hr. LDL solution was then dialyzed at 4° C. against0.15M NaCl/0.3 mM EDTA for 2 days with several changes, before beingremoved and concentrated using an Amicon spin column by spinning for 1hr. 4000 RPM at 4° C.

[0345] For acetylation, 1 ml of 5 mg/ml LDL was added to 1 ml of asaturated solution of NaOAc in a 15 ml tube on ice on a shaker at 4° C.8 μl of acetic anhydride was added 2 μl at a time over 1 hr. LDL wasthen dialyzed for 48 hr. against 0.15M NaCl/0.3 mM EDTA at 4° C. for 48hr. with several changes. Final concentrations of derivatized LDL's weredetermined by comparing to a dilution curve of native LDL analyzed atOD₂₈₀, with 0.15M NaCl/0.3 mM EDTA used as diluent in all cases.

[0346] 6.1.2. Analysis of Paradigm Material

Differential Display

[0347] Removal of DNA:

[0348] The RNA pellet was resuspended in H₂O and quantified byspectrophotometry at OD₂₆₀. Approximately half of the sample was thentreated with DNAse I to remove contaminating chromosomal DNA. RNA wasamplified by PCR using the following procedure. 50 ul RNA sample (10-20μg), 5.7 μl 10× PCR buffer (Perkin-Elmer/Cetus), 1 μl RNAse inhibitor(40 units/μl) (Boehringer Mannheim, Germany) were mixed together,vortexed, and briefly spun. 2 μl DNAse I (10 units/μl) (BoehringerMannheim) was added to the reaction which was incubated for 30 min. at37° C. The total volume was brought to 200 μl with DEPC H₂O, extractedonce with phenol/chloroform, once with chloroform, and precipitated byadding 20 μl 3M NaOAc, pH 4.8, (DEPC-treated), 500 μl absolute ETOH andincubating for 1 hour on dry ice or −20° C. overnight. The precipitatedsample was centrifuged for 15 min., and the pellet was washed with 70%ETOH. The sample was re-centrifuged, the remaining liquid was aspirated,and the pellet was resuspended in 100 μl H₂O. The concentration of RNAwas measured by reading the OD₂₆₀.

[0349] First Strand cDNA Synthesis:

[0350] For each RNA sample duplicate reactions were carried out inparallel. 400 ng RNA plus DEPC H₂O in a total volume of 10 μl were addedto 4 μl T₁₁XX reverse primer (10 μM) (Operon). The specific primers usedin each experiment are provided in the Description of the Figures inSection 4, above. The mixture was incubated at 70° C. for 5 min. todenature the RNA and then placed at r.t. 26 μl of reaction mixcontaining the following components was added to each denaturedRNA/primer sample: 8 μl 5× First Strand Buffer (Gibco/BRL, Gaithersburg,Md.), 4 μl 0.1M DTT (Gibco/BRL), 2 μl RNAse inhibitor (40 units/μl)(Boehringer Mannheim), 4 μl 200 μM dNTP mix, 6 μl H₂O, 2 μl Superscriptreverse transcriptase (200 units/μl) (Gibco/BRL). The reactions weremixed gently and incubated for 30 min. at 42° C. 60 μl of H₂O (finalvolume=100 μl) were then added and the samples were denatured for 5 min.at 85° C. and stored at −20° C.

[0351] PCR Reactions:

[0352] 13 μl of reaction mix was added to each 5tube of a 96 well plateon ice. The reaction mix contained 6.4 μl H₂O, 2 μl 10× PCR Buffer(Perkin-Elmer), 2 μl 20 μM dNTP's, 0.4 μl ³⁵S dATP (12.5 μCi/μl; 50 μCitotal) (Dupont/NEN), 2 μl forward primer (10 μM) (Operon), and 0.2 μlAmpliTaq Polymerase (5 units/μl) (Perkin-Elmer). Next, 2 μl of reverseprimer (T₁₁XX, 10 μM) were added to the side of each tube followed by 5μl of cDNA also to the sides of the tubes, which were still on ice. Thespecific primets used in each experiment are provided in the Descriptionof the Figures in Section 4, above. Tubes were capped and mixed, andbrought up to 1000 RPM in a centrifuge then returned immediately to ice.The PCR machine (Perkin-Elmer 9600) was programmed for differentialdisplay as follows: $*={{X40}\begin{matrix}{94{^\circ}\quad {C.}} & {\quad {2\quad {\min.}}} \\{*94{^\circ}\quad {C.}} & {\quad {15\quad {\sec.}}} \\{*40{^\circ}\quad {C.}} & {\quad {2\quad {\min.}}} \\{*{ramp}\quad 72{^\circ}\quad {C.}} & {\quad {72{^\circ}\quad {C.\quad 1}\quad {\min.}}} \\{*72{^\circ}\quad {C.}} & {\quad {30\quad {\sec.}}} \\{72{^\circ}\quad {C.}} & {\quad {5\quad {\min.}}} \\{4{^\circ}\quad {C.}} & {\quad {hold}}\end{matrix}}$

[0353] When the PCR machine reached 94° C., the plate was removed fromice and placed directly into the Perkin-Elmer 9600 PCR machine.Following PCR, 15 μl of loading dye, containing 80% formamide, 10 mMEDTA, 1 mg/ml xylene cyanol, 1 mg/ml bromphenol blue were added. Theloading dye and reaction were mixed, incubated at 85° C. for 5 min.,cooled on ice, centrifuged, and placed on ice. Approximately 4 μl fromeach tube were loaded onto a prerun (60V) 6% acrylamide gel. The gel wasrun at approximately 80V until top dye front was about 1 inch frombottom. The gel was transferred to 3MM paper (Wha-tman Paper, England)and dried under vacuum. Bands were visualized by autoradiography.

[0354] Band Isolation and Amplification:

[0355] Differentially expressed bands were excised from the dried gelwith a razor blade and placed into a microfuge tube with 100 μl H₂O andheated at 100° C. for 5 min., vortexed, heated again to 100° C. for 5min., and vortex again. After cooling, 100 μl H₂O, 20 μl 3M NaOAc, 1 μlglycogen (20 mg/ml), and 500 μl ethanol were added and chilled. Aftercentrifugation, the pellet was washed and resuspended in 10 μl H₂O.

[0356] The isolated differentially expressed bands were then amplifiedby PCR using the following reaction conditions: 58 μl H₂O 10 μl 10 x PCRBuffer 10 μl 200 μm dNTP's 10 μl 10 μM reverse primer 10 μl 10 μMforward primer 1.5 μl amplified band 0.5 μl AmpliTaq polymerase (5units/μl) (Perkin Elmer)

[0357] PCR was performed using the program described in this Section,above, for differential display. After PCR, glycerol loading dyes wereadded and samples were loaded onto a 2% preparative TAE/Biogel (Bio101,La Jolla, Calif.) agarose gel and eluted. Bands were then excised fromthe gel with a razor blade and vortexed for 15 min. at r.t., andpurified using the Mermaid kit from Bio101 by adding 3 volumes ofMermaid high salt binding solution and 8 μl of resuspended glassfog in amicrofuge tube. Glassfog was then pelleted, washed 3 times with ethanolwash solution, and then DNA was eluted twice in 10 μl at 50° C.

[0358] Subcloning:

[0359] The TA cloning kit (Invitrogen, San Diego, Calif.) was used tosubclone the amplified bands. The ligation reaction typically consistedof 4 μl sterile H₂O, 1 μl ligation buffer, 2 μl TA cloning vector, 2 μlPCR product, and 1 μl T4 DNA ligase. The volume of PCR product can vary,but the total volume of PCR product plus H₂O was always 6 μl. Ligations(including vector alone) were incubated overnight at 12° C. beforebacterial transformation. TA cloning kit competent bacteria (INVαF′:enda1, recAl, hsdR17(r−k, m+k), supE44, λ-, thi-1, gyrA, relA1,φ80lacZαΔM15Δ(1acZYA-argF), deoR+, F′) were thawed on ice and 2 μl of0.5 M β-mercaptoethanol were added to each tube. 2 μl from each ligationwere added to each tube of competent cells (50 μl), mixed withoutvortexing, and incubated on ice for 30 min. Tubes were then placed in42° C. bath for exactly 30 sec., before being returned to ice for 2 min.450 μl of SOC media (Sambrook et al., 1989, supra) were then added toeach tube which were then shaken at 37° C. for 1 hr. Bacteria were thenpelleted, resuspended in ˜200 μl SOC and plated on Luria broth agarplates containing X-gal and 60 μg/ml ampicillin and incubated overnightat 37° C. White colonies were then picked and screened for inserts usingPCR.

[0360] A master mix containing 2 μl 10× PCR buffer, 1.6 μl 2.5 mMdNTP's, 0.1 μl 25 mM MgCl₂, 0.2 μl M13 reverse primer (100 ng/μl), 0.2μl M13 forward primer (100 ng/pl), 0.1 μl AmpliTaq (Perkin-Elmer), and15.8 μl H₂O was made. 40 μl of the master mix were aliquoted into tubesof a 96 well plate, and whole bacteria were added with a pipette tipprior to PCR. The PCR machine (Perkin-Elmer 9600) was programmed forinsert screening as follows: $*={{X35}\begin{matrix}{94{^\circ}\quad {C.}} & {\quad {2\quad {\min.}}} \\{*94{^\circ}\quad {C.}} & {\quad {15\quad {\sec.}}} \\{*47{^\circ}\quad {C.}} & {\quad {2\quad {\min.}}} \\{*{ramp}\quad 72{^\circ}\quad {C.}} & {\quad {72{^\circ}\quad {C.\quad 30}\quad {\sec.}}} \\{*72{^\circ}\quad {C.}} & {\quad {30\quad {\sec.}}} \\{72{^\circ}\quad {C.}} & {\quad {10\quad {\min.}}} \\{4{^\circ}\quad {C.}} & {\quad {hold}}\end{matrix}}$

[0361] Reaction products were eluted on a 2% agarose gel and compared tovector control. Colonies with vectors containing inserts were purifiedby streaking onto LB/Amp plates. Vectors were isolated from such strainsand subjected to sequence analysis, using an Applied BiosystemsAutomated Sequencer-(Applied Biosystems, Inc. Seattle, Wash.).

[0362] Northern Analysis:

[0363] Northern analysis was performed to confirm the differentialexpression of the genes corresponding to the amplified bands. The probesused to detect mRNA were synthesized as follows: typically 2 Alamplified band (˜30 ng), 7 μl H₂O, and 2 μl 10× Hexanucleotide mix(Boehringer-Mannheim) were mixed and heated to 95° C. for 5 min., andthen allowed to cool on ice. The volume of the amplified band can vary,but the total volume of the band plus H₂O was always 9 μl. 3 μldATP/dGTP/dTTP mix (1:1:1 of 0.5 mM each), 5 μl α³²P dCTP 3000 Ci/MM (50μCi total) (Amersham, Arlington Heights, Ill.), and 1 μl Klenow (2units) (Boehringer-Mannheim) were mixed and incubated at 37° C. After 1hr., 30 μl TE were added and the reaction was loaded onto a Biospin-6™column (Biorad, Hercules, Calif.), and centrifuged. A 1 μl aliquot ofeluate was used to measure incorporation in a scintillation counter withscintillant to ensure that 10⁶cpm/μl of incorporation was achieved.

[0364] The samples were loaded onto a denaturing agarose gel. A 300 ml1% gel was made by adding 3 g of agarose (SeaKem™ LE, FMC BioProducts,Rockland, Me.) and 60 ml of 5× MOPS buffer to 210 ml sterile H₂O. 5×MOPS buffer (0.1M MOPS (pH 7.0), 40 mM NaOAc, 5 mM EDTA (pH 8.0)) wasmade by adding 20.6 g of MOPS to 800 ml of 50 mM NaOAc (13.3 ml of 3MNaOAc pH 4.8 in 800 ml sterile H₂O); then adjusting the pH to 7.0 with10M NaOH; adding 10 ml of 0.5M EDTA (pH8.0); and adding H₂O to a finalvolume of 1L. The mixture was heated until melted, then cooled to 50°C., at which time 5 μl ethidium bromide (5mg/ml) and 30 ml of 37%formaldehyde of gel were added. The gel was swirled quickly to mix, andthen poured immediately.

[0365] 2 μg RNA sample, lx final 1.5× RNA loading dyes (60% formamide,9% formaldehyde, 1.5× MOPS, 0.075% XC/BPB dyes) and H₂O were mixed to afinal volume of 40 μl. The tubes were heated at 65° C. for 5 min. andthen cooled on ice. 10 μg of RNA MW standards (New England Biolabs,Beverly, Mass.) were also denatured-with dye and loaded onto the gel.The gel was run overnight at 32V in MOPS running buffer.

[0366] The gel was then soaked in 0.5 μg/ml Ethidium Bromide for 45min., 50 mM NaOH/0.1 M NaCl for 30 min., 0.1 M Tris pH 8.0 for 30 min.,and 20× SSC for 20 min. Each soaking step was done at r.t. with shaking.The gel was then photographed along with a fluorescent ruler beforeblotting with Hybond-N membrane (Amersham), according to the methods ofSambrook et al., 1989, supra, in 20× SSC overnight.

[0367] For hybridization, the blot was placed into a roller bottlecontaining 10 ml of prehybridization solution consisting of 50%formamide and 1× Denhardt's solution, and placed into 65° C. incubatorfor 30 min. The probe was then heated to 95° C., chilled on ice, andadded to 10 ml of hybridization solution, consisting of 50% formamide,1× Denhardt's solution, 10% dextransulfate, to a final concentration of10⁶ cpm/ml. The prehybridization solution was then replaced with theprobe solution and incubated overnight at 42° C. The following day, theblot was washed three times for 30 min. in 2× SSC/0.1% SDS at roomtemperature before being covered in plastic wrap and put down forexposure.

[0368] RT-PCR Analysis:

[0369] RT-PCR was performed to detect differentially expressed levels ofmRNA from the genes corresponding to amplified bands. First strandsynthesis was conducted by mixing 20 μl DNased RNA (˜2 μg), 1 μl oligodT (Operon) (1 μg), and 9.75 μl H₂O. The samples were heated at 70° C.for 10 min., and then allowed to cool on ice. 10 μl first strand buffer(Gibco/BRL), 5 μl 0.1M DTT, 1.25 μl 20 mM dNTP's (500 μM final), 1 μlRNAsin (40 units/μl) (Boehringer Mannheim), and 2 μl Superscript ReverseTranscriptase (200 units/μl) (Gibco/BRL) were added to the reaction,incubated at 42° C. for 1 hr., and then placed at 85° C. for 5 min., andstored at −20° C.

[0370] PCR was performed on the reverse transcribed samples. Eachreaction contained 2 μl 10× PCR buffer, 14.5 μl H₂O, 0.2 μl 20 mM dNTP's(200 μM final), 0.5 μl 20 μM forward primer (0.4 μM final), 0.5 μl 20 μMreverse primer (0.4 μM final), 0.3 μl AmpliTaq polymerase(Perkin-Elmer/Cetus), 2 μl cDNA dilution or positive control (40 pg).The specific primers used in each experiment are provided in theDescription of the Figures in Section 4, above. Samples were placed inthe PCR 9600 machine at 94° C. (hot start), which was programmed asfollows: $*={35x\begin{matrix}{94{^\circ}\quad {C.}} & {\quad {2\quad {\min.\quad \left( {{samples}\quad {loaded}} \right)}}} \\{*94{^\circ}\quad {C.}} & {\quad {45\quad {\sec.}}} \\{*55{^\circ}\quad {C.}} & {\quad {45\quad {\sec.}}} \\{*72{^\circ}\quad {C.}} & {\quad {2\quad {\min.}}} \\{\quad {72{^\circ}\quad {C.}}} & {\quad {5\quad {\min.}}} \\{4{^\circ}\quad {C.}} & {\quad {hold}}\end{matrix}}$

[0371] Reactions were carried out on cDNA dilution series and tubes wereremoved at various cycles from the machine during 72° C. step. Reactionproducts were eluted on a 1.8% agarose gel and visualized with ethidiumbromide.

[0372] 6.1.3. Chromosomal Localization of Target Genes

[0373] Once the nucleotide sequence has been determined, the presence ofthe gene on a particular chromosome is detected. Oligonucleotide primersbased on the nucleotide sequence of the target gene are used in PCRreactions using individual human chromosomes as templates. Individualsamples of each the twenty-three human chromosomes are commerciallyavailable (Coriel Institute for Medical Research, Camden, N.J.). Thechromosomal DNA is amplified according to the following conditions: longchromosomal DNA, 2μl 10× PCR buffer, 1.6μl 2.5 mM dNTP's, 0.1 μl 25 mMMgCl₂, 0.2 μl reverse primer (100ng/μl), 0.2μl forward primer (100ng/μl), 0.1 μl Taq polymerase, and 15.8μl H₂O. Samples are placed in thePCR 9600 machine at 94° C. (hot start), which is programmed as follows:$*={35x\begin{matrix}{94{^\circ}\quad {C.}} & {\quad {2\quad {\min.\quad \left( {{samples}\quad {loaded}} \right)}}} \\{*94{^\circ}\quad {C.}} & {\quad {20\quad {\sec.}}} \\{*55{^\circ}\quad {C.}} & {\quad {30\quad {\sec.}}} \\{*72{^\circ}\quad {C.}} & {\quad {30\quad {\sec.}}} \\{\quad {72{^\circ}\quad {C.}}} & {\quad {5\quad {\min.}}} \\{4{^\circ}\quad {C.}} & {\quad {hold}}\end{matrix}}$

7. EXAMPLE: IDENTIFICATION OF GENES DIFFERENTIALLY EXPRESSED IN RESPONSETO PARADIGM B: IN VIVO MONOCYTES

[0374] In an alternative embodiment of the invention, genesdifferentially expressed in monocytes were detected under highlyphysiologically relevant, in vivo conditions. According to Paradigm B,human subjects were held in a clinical setting and the fat/cholesterolcontent of their diets was controlled. Monocytes were isolated atdifferent stages of treatment, and their gene expression pattern wascompared to that of control groups.

[0375] By use of Paradigm B, the human bcl-2 gene was identified. Itsexpression decreases in response to the atherogenic conditions of highfat/high cholesterol (FIG. 1). The Apo E−/− mouse is the first mousemodel of atherosclerosis with pathology similar to that of human plaquedevelopment (Plump et al., 1992, Cell 71: 343-353). Serum cholesterollevels in these mice on a chow diet is five times higher than those ofcontrol littermates. To address whether the regulation of the mousebcl-2 gene is also affected by serum cholesterol levels, monocytes fromapoE-deficient mice and littermate wild-type controls were purified andmouse bcl-2 mRNA levels were compared using quantitative RT-PCR. By thismethod, mouse bcl-2 MRNA levels were significantly lower in theapoE-deficient mice relative to the wild-type controls (FIG. 3).

[0376] The differential expression pattern of the human glutathioneperoxidase gene (HUMGPXP1) was also discovered. The differentialexpression of HUMGPXP1 was initially detected in a preliminary detectionsystem, described, below, in Section 7.1.2. Once HUMGPXP1 sequences wereobtained, the gene's differential expression pattern was verified andcharacterized under the physiologically relevant conditions of ParadigmB. Glutathione peroxidase is known to be involved in the removal oftoxic peroxides that form in the course of growth and metabolism undernormal aerobic conditions and under oxidative stress. Human plasmaglutathione peroxidase gene was originally isolated from a humanplacenta cDNA library (Takahashi et al., 1990, J. Biochem. 108:145-148). It has been shown to be expressed in two human cell lines ofthe myeloid lineage (Porter et al., 1992, Clinical Science 83: 343-345).Other studies have also linked reduced levels of this enzyme with heartattack risk (Guidi, et al., 1986, J. Clin. Lab Invest. 46: 549-551; Wanget al., 1981, Klin. Wochenschr. 59: 817-818; Kok et al., 1989, J. Am.Med. Assoc. 261: 1161-1164; and Gromadzinska & Sklodowska, 1990, J. Am.Med. Assoc. 263: 949-950). Glutathione peroxidase has not beenpreviously known to be down-regulated in human monocytes undercardiovascular disease conditions, as described herein.

[0377] Interestingly, bcl-2 has been recognized as playing a key role inpreventing apoptosis, and expression of glutathione peroxidase in theabsence of bcl-2 is able to compensate for this loss by preventingapoptosis (Hockenbery et al., 1993, Cell 75: 241-251). These findingsregarding bcl-2 and HUMGPXP1, described herein in this section,suggested a novel role for the monocyte in plaque formation whichinvolves apoptosis induction caused by high LDL concentrations insidethe cell, or perhaps by oxidative stress in the cell mediated byoxidized LDL.

[0378] To confirm this relationship between apoptosis andatherosclerosis, the ability of bcl-2 expression to ameliorateatherosclerosis is tested. Because bcl-2 is normally down-regulatedunder atherogenic conditions, a transgenic mouse strain is engineered inwhich the human bcl-2 gene is expressed under the control of thescavenger receptor promoter, which is induced in monocyte foam cellsunder atherogenic conditions. This transgenic mouse is then crossed withan apoE-deficient atherosclerotic mouse model. The ability of theincreased expression of the bcl-2 target gene to ameliorateatherosclerosis is demonstrated by a decrease in initiation andprogression of plaque formation observed in the transgenicapoE-deficient mouse.

[0379] The identification of the differential expression of these genes,therefore, provides targets for the treatment and diagnosis ofcardiovascular disease. Intervening in the apoptotic pathway throughBcl-2 and glutathione peroxidase, may lead to lesion regression orprevention of plaque formation, or both. Furthermore, the discovery of aconnection between the apoptotic pathway and atherosclerosisdemonstrates the effectiveness of the methods described herein inidentifying the full panoply of gene products that are involved in theatherosclerotic disease process. Furthermore, the down-regulation ofbcl-2 and HUMGPXP1 under Paradigm B provides a fingerprint for the studyof the effect of excess LDL on monocytes.

[0380] 7.1. Materials and Methods

[0381] 7.1.1. in vivo Cholesterol Studies

[0382] Patients were held in a clinical setting for a total of 9 weeksduring which time their lipid intake was very tightly controlled. Therewere a total of 3 diets, and each patient was held on each diet for 3weeks. Patients were healthy young (third decade of life) individualswith no history or symptoms of heart disease or dislipidemias. The 3diets are described below: American Heart Association Diet II fat 25%cholesterol  80 mg/1000 kCal polyunsaturated/saturated fat 1.5 AverageAmerican Diet fat 43% cholesterol 200 mg/1000 kCalpolyunsaturated/saturated fat 0.34 Combination Diet fat 43% cholesterol 80 mg/1000 kCal polyunsaturated/saturated fat 0.34

[0383] The 3 diets were isocaloric, and the individual components ofeach diet may vary with the participant's preference as long as thelipid levels in the diet were maintained.

[0384] Cell Isolation

[0385] At the end of each 3 week diet period, blood was drawn from eachpatient after a 12 hour period of fasting and monocytes were purified.50 ml of blood was drawn into 5 evacuated tubes containing 1.4 ml eachof citrate phosphate dextrose to prevent coagulation. Blood was pooledinto 50 ml tubes and spun at 400 g (1250 RPM/Sorvall RC3B) for 15minutes at 4° C. The upper serum layer (˜25 ml) was then removed with apipette and replaced with phosphate buffered saline (PBS) at 4° C. Theblood was mixed and then spun at 1850 × g (2680 RPM) for 15 minutes at4° C. Most of the clear upper layer was removed with a pipette, beforethe buffy coat at the interface was taken in ˜5 ml. The buffy coat wasplaced into a separate 50 ml tube, and the pipette used to remove it waswashed with 20 ml PBS. A small aliquot of these cells was then diluted1:1000 in PBS and counted under a microscope using a hemacytometer. Redblood cell concentration was then adjusted with PBS to a finalconcentration of 1.5×10⁹/ml, and 10 ml aliquots were added to LeucoprepBecton Dickinson) tubes for monocyte isolation. Tubes were spun for 25minutes at 25° C. in a Sorvall RT6000 with the brake off. Most of theclear upper layer was discarded, and the turbid layer above the gel wassaved and pooled in 50 ml tubes. The volume of each tube was thenincreased to 50 ml with 25° C. PBS, and spun at 1000 RPM (Sorvall RC3B)for 10 minutes at 4° C. The liquid was then discarded, the pellet wasresuspended in 50 ml PBS, and spun again. This process was repeated 3more times. The final cell pellet was then resuspended in 2 ml RNA lysisbuffer (Sambrook et al., 1989, supra) and frozen for subsequent RNAisolation as described above in Section 6.1.1.

[0386] Differential display, Northern analysis, RT-PCR, subcloning, andDNA sequencing were carried out as described, above, in Section 6.1.2.

[0387] 7.1.2. Preliminary Detection System

[0388] The preliminary detection system described in this section wasused to identify sequences that are differentially expressed in areadily assayed, in vitro system. Sequences that showed some homology tothose thought to be involved in cardiovascular disease were then used asspecific primers or probes, or both, in Paradigm B, wherein thedifferential expression was ascertained under physiologically relevantconditions, as described in section 7.1.1, above.

[0389] Cell Culture

[0390] Blood (˜100 ml) was drawn from healthy human donors intovacutainer tubes containing heparin (Becton Dickinson). Blood wasdiluted 1:1 with PD (Phosphate buffered saline (PBS) without Ca or Mg,plus 0.3 mM EDTA), and layered onto Ficoll (Lymphocyte SeparationMedia—Organon Teknikon) as 30 ml of blood/7 ml ficoll in a 50 mlblue-capped Falcon tube, and centrifuged at 2000 RPM for 25 min. at roomtemperature (r.t.). The buffy coat was removed with a pipette,transferred to another 50 ml tube, diluted to 30 ml with PD, andcentrifuged at 1200 RPM for 10 min. at r.t. The pellet was resuspendedin 30 ml PD and the previous centrifugation step was repeated. Thepellet was resuspended in 40 ml RPMI (2 mM1-Glutamine+penicillin/streptomycin), plated onto 4 plates, andincubated at 37° C. for 2 hours. Supernatant was removed, and the plateswere washed 3× with PBS at 37° C. Plates were finally resuspended in 10ml each with RPMI/20% human AB serum (Sigma, St. Louis, MO.). On day 5,the media was changed and 100 units/ml of human γ-IFN (Genzyme) wereadded. On day 7, the media was removed and replaced with RPMI/20% humanLDL-deficient serum +100 units/ml of human γ-IFN. Native, oxidized, andacetylated LDL were each added to one plate with the fourth plateserving as control. After the specified incubation time (5 hr. or 24hr.) the media was removed and the cells were resuspended in 2 mlguanidine isothiocyanate RNA lysis buffer (Sambrook et al., 1989,supra). Lysed cells were then syringed with 23 G. needle, layered over5.7M CsCl, and centrifuged for 20 hr. at 35K RPM. RNA was isolatedaccording to the method of Sambrook et al., 1989, supra.

[0391] Lipoproteins were prepared as described, above, in section 6.1.1.Differential display, Northern analysis, RT-PCR, subcloning, and DNAsequencing were carried out as described, above, in Section 6.1.2. Fordifferential display, the primers used were T₁₁CC (reverse) and OPE4(forward), consisting of 5′GTGACATGCC3′. For RT-PCR, the first strandcDNA was primed with T₁₁CC, and PCR reactions were carried out withrfhma15 primers (for-catgcctgtagaaaaaggtt/rev-cttcatagaatctaagccta), andmouse γactin primers(for-cctgatagatgggcactgtgt/rev-gaacacggcattgtcactaact).

[0392] 7.1.3. Transgenic ApoE-Deficient Mouse Expressing Human bcl-2

[0393] Transgenic mice bearing a construct (FIG. 32) with the mousescavenger receptor regulatory element (5 kb) (M. Freeman, et al., 1995,unpublished results) driving expression of the human bcl-2 gene (hbcl-2)were produced. The scavenger receptor regulatory element (ScR) is knownto activate reporter gene expression in peritoneal macrophages intransgenic mice (M. Freeman, 1995, unpublished results). This 5 kbfragment is linked to the human bcl-2 cDNA (Cleary, et al., 1986, supra)via a NotI restriction site. Human growth hormone (hGH) sequences (Mayo,et al., 1983, Nature 306: 86-88) are then ligated onto the 3′ end ofthis Construct through filled-in BamHI and EcoRV sites to providemessage stability. This construct is then digested with XhoI and the 9kb ScR-hbcl2-hGH sequences are purified away from vector sequences.Another plasmid sample is digested with KpnI to yield a fragment withonly 1.5 kb of scavenger receptor regulatory sequences which provide alower level of expression. These fragments are then injectedindependently into mouse embryos derived from the FVB and C57BL/6 mousestrains according to standard protocols (Hogan, et al., Manipulating theMouse Embryo, 1994, Cold Spring Harbor Laboratory Press). Followingbirth, tail sections are cut from mice derived from injected embryos andanalyzed for the presence of transgene sequences using hbcl-2 sequencesas probes on Southern blots.

[0394] Transgenic mice bearing the ScR-hbcl2-hGH construct are then bredto wild-type mice of the same respective strain, and then the offspringare backcrossed to produce homozygous lines of mice. These mice are thenbred to apoE-deficient mice. offspring are analyzed for presence of theScR-hbcl2-hGH by preparing tail sections and probing with hbcl-2sequences on Southern blots. Offspring are then analyzed for lesionformation and progression according to the methods of Plump, et al.,1992, supra.

[0395] 7.2. Results

[0396] Differential display analysis was carried out on monocyte RNAderived from the blood of patients whose serum cholesterol levels weremanipulated through fat/cholesterol intake in their diets. FIG. 1 showsband #14 which was present in the low dietary fat/low serum cholesterolconditions and goes away in the high dietary fat/high serum cholesterolconditions. When a radioactively labeled probe was prepared from band#14 and hybridized with a Northern blot prepared from RNA from the samepatient (FIG. 2), an 8 kb band was seen which was present in low serumcholesterol and disappeared in high serum cholesterol conditions. Whenband #14 sequences were subcloned, sequenced, and compared with thesequence database a 98% (203/207 bp) sequence similarity with the humanbcl-2 gene (Cleary et al., 1986, Cell 47, 19-28) was obtained,indicating that band #14 is bcl-2.

[0397] Glutathione peroxidase (HUMGPXP1) in expression in monocytes wasexamined to determine its physiological relationship to bcl-2.Differential expression of HUMGPXP1 was first detected in a preliminarydetection system using monocytes cultured in vitro. Human monocytes wereprepared as described above in subsection 7.1.2. Cells were lysed after5 hours and RNA was prepared. Differential display analysis was carriedout, and regulated bands were isolated and characterized. The DNAsequence was determined from a number of independent subclones ofamplified sequences of one such regulated band designated band 15. Usingthe BLAST program (Altschul, et al., 1990, J. Mol. Biol. 215: 403-410),a 176/177 (99%) sequence similarity was found between band 15 a sequencefor human plasma glutathione peroxidase exon 1 (HUMGPXP1). This sequenceoccurs upstream of the reported transcription start site. Nonetheless,RT-PCR analysis confirmed that the band 15 sequences are in fact withinthe same transcription unit as sequences downstream of the reportedtranscription start site.

[0398] Based on this preliminary result, the gene expression pattern ofglutathione peroxidase (HUMGPXP1) was further analyzed for verificationand characterization in physiologically relevant samples according toParadigm B. Monocytes derived from human blood under atherogenicconditions (high serum cholesterol) and healthy conditions (low serumcholesterol) were examined with RT-PCR. As shown in FIG. 4, thereappears to be 2-3 fold less cDNA amplified by the HUMGPXP1 primers fromthe high fat/cholesterol monocytes than in the low fat/cholesterolmonocytes, while the actin control bands are the same.

[0399] Monocytes from apoE-deficient mice and littermate wild-typecontrols were purified and mouse bcl-2 mRNA levels were compared usingquantitative RT-PCR. By this method, mouse bcl-2 mRNA levels weresignificantly lower in the apoE-deficient mice relative to the wild-typecontrols (FIG. 3).

[0400] These results demonstrate that bcl-2 is an excellent target genefor intervening in lesion formation and development. It was previouslyknown that, under normal conditions, bcl-2 expression preventsapoptosis. The observed down-regulation of bcl-2 caused by atherogenicconditions, therefore, provides an explanation of how such atherogenicconditions may lead to plaque formation. By down-regulating the normallyprotective bcl-2 gene, high serum cholesterol triggers a series ofevents, entailing the induction of the apoptotic pathway, which resultsin programmed cell death, which in turn causes an inflammatory responseand subsequent plaque formation.

[0401] This model may be tested by counteracting the observeddown-regulation of bcl-2. The human bcl-2 gene is placed in theScR-hbcl2-hGH construct in which it is transcribed by a promoter that isactivated in monocyte foam cells under atherogenic conditions. Thisconstruct is then introduced into an apoE-deficient mouse that otherwiseserves as a model for atherosclerosis. The effect of bcl-2 expression onatherosclerosis is evidenced by the reduction in plaque initiation anddevelopment in the apoE-deficient mice bearing the construct.Amelioration of atherosclerosis may, therefore, be accomplished by suchintervention in the down-regulation of the bcl-2 target gene.

8. EXAMPLE: IDENTIFICATION OF GENES DIFFERENTIALLY EXPRESSED IN RESPONSETO PARADIGM C: IL-1 INDUCTION OF ENDOTHELIAL CELLS

[0402] According to the invention, differential display was used todetect four novel genes that are differentially expressed in endothelialcells that were treated in vitro with IL-1. Three of these genes,rchdO24, rchdO32, and rchdO36, are not homologous to any known gene. Thefourth gene, rchdoo5, is 70% homologous to a cloned shark gene calledbumetanide-sensitive Na—K—Cl cotransport protein. A human homolog ofthis gene has been reported, but the sequence has not yet been published(1994, Proc. Natl. Acad. Sci. USA 91: 2201-2205). The discovery of theup-regulation of these four genes provides a fingerprint profile of IL-1induced endothelial cells. This fingerprint profile can be used in thetreatment and diagnosis of cardiovascular iseases, including but notlimited to atherosclerosis, ischemia/reperfusion, hypertension,restenosis, and arterial inflammation.

[0403] 8.1. Materials and Methods

[0404] Primary cultures of HUVEC's were established from normal termumbilical cords as described (In Progress in Hemostasis and Thrombosis,Vol. 3, P. Spaet, editor, Grune & Stratton Inc., New York, 1-28). Cellswere grown in 20% fetal calf serum complete media (1989, J. Immunol.142: 2257-2263) and passaged 1-3 times before activation.

[0405] For activation, cells were cultured with 10 units/ml of humanIL-1β for 1 or 6 hr. before lysis in guanidinium isothiocyanate RNAlysis buffer (Sambrook et al., 1989, supra). Lysed cells were thensyringed with a 23 G. needle, layered over 5.7M CsCl, and centrifugedfor 20 hr. at 35K.

[0406] Alternatively, cells were induced in the presence of 100 μMlysophosphatidylcholine, or 50 μg/ml oxidized human LDL (Sigma) forperiods of 1 or 6 hr. RNA was isolated as described, above, in Section6.1. Differential display, Northern analysis, RT-PCR, subcloning, andDNA sequencing were carried out as described, above, in Section 6.1.2,except that Northern blot hybridizations were carried out as follows:for pre-hybridization, the blot was placed into roller bottle containing10 ml of rapid-hyb solution (Amersham), and placed into 65° C. incubatorfor at least 1 hr. For hybridization, 1×10⁷ cpm of the probe was thenheated to 95° C., chilled on ice, and added to 10 ml of rapid-hybsolution. The prehybridization solution was then replaced with probesolution and incubated for 3 hr at 65° C. The following day, the blotwas washed once for 20 min. at r.t. in 2× SSC/0.1% SDS and twice for 15min. at 65° C. in 0.1× SSC/0.1% SDS before being covered in plastic wrapand put down for exposure.

[0407] Chromosomal locations were determined according to the methoddescribed in Section 6.1.3, above. For rchdO24, the primers used werefor-cccatagactaggctcatag, and rev-tttaaagagaaattcaaatc.

[0408] 8.2. Results

[0409] HUVEC's were activated with 10 units/ml IL-1β for 1 or 6 hoursand compared to resting HUVEC's using differential display. As shown inFIG. 5, a band marked rchd005 is present in lanes 11 and 12 (IL-1, 6hr.) but not in lanes 9 and 10 (control), or lanes 7 and 8 (IL-1, 1hr.). This band, rchd005, was isolated and subcloned and sequenced. Whena probe prepared form this band was used to screen a Northern blot,expression was seen at 6 hr., but not at 1 hr. or in the control (FIG.6). However, when this same probe was hybridized to a Northern blotprepared from shear stressed RNA, according to Paradigm D described inSection 9, below, a different pattern of up-regulation was also seen(FIG. 7). Expression was up at 1 hr. and then nearly disappeared by 6hr. Amplified rchdOo5 DNA was subcloned and sequenced. Sequence analysisrevealed an approximately 360 bp insert (FIG. 8) with 70% sequencesimilarity to a cloned shark gene called bumetanide-sensitive Na—K—Clcotransport protein.

[0410] Another IL-1 inducible band, rchd024, is shown in FIG. 9.Northern analysis on IL-1 up-regulated RNA reveals a kb message presentat 6 hr. (FIG. 10) that also shows a low level of up-regulation undershear stress at 6 hr. (FIG. 11). The DNA sequence was obtained fromsubclones of amplified DNA (FIG. 12). Database searching revealed nosignificant sequence similarities. A PCR amplification experimentdetermined that the rchd024 gene is located on human chromosome 4.

[0411] Band rchd032 was isolated on the basis of its differentiallyincreased expression after 6 hr. treatment with IL-1 (FIG. 13), whichwas confirmed by RT-PCR analysis (FIG. 14). Amplified rchd032 sequenceswere subcloned and sequenced (FIG. 15). No significant homology to anyknown gene was found.

[0412] Band rchd036 was also isolated on the basis of its differentialexpression 6 hr. after IL-1 treatment (FIG. 16). Northern analysis (FIG.17) revealed an 8 kb band which was up-regulated 6 hr. after IL-1treatment. Another Northern analysis was performed testing rchd036 underthe shear stress condition of Paradigm D, which are described in theexample in Section 9, below. Interestingly, rchd036 is not induced byshear stress, as indicated by the lack of any band after either 1 hr. or6 hr. of treatment (FIG. 33). This tesult provides an example of anIL-1-inducible endothelial cell gene that is not regulated by shearstress, indicating that these induction pathways can be separated, andmay provide for drugs with greater specificity for the treatment ofinflammation and atherosclerosis. The DNA sequence was obtained fromsubclones of amplified DNA (FIG. 18), and a search of the databaserevealed no sequence similarities. A PCR amplification experimentdetermined that the rchd036 gene is located on human chromosome 15.

9. EXAMPLE: IDENTIFICATION OF GENES DIFFERENTIALLY EXPRESSED IN RESPONSETO PARADIGM D: ENDOTHELIAL CELL SHEAR STRESS

[0413] According to the invention, differential display was used todetect genes that are differentially expressed in endothelial cells thatwere subjected to fluid shear stress in vitro. Shear stress is thoughtto be responsible for the prevalence of atherosclerotic lesions in areasof unusual circulatory flow. Using the method of Paradigm D, four bandswith novel DNA sequences were identified. rchd528 does not sharehomology with any known gene. rchd502, on the other hand is homologousto rat matrin F/G mRNA sequence. This rat gene encodes a protein that isfound in the nuclear matrix and contains the zinc finger DNA bindingmotif, (Hakes, et al., 1991, Proc. Natl. Acad. Sci. U.S.A.88:6186-6190). In fact, the sequences in rchd502 encode part of the zincfinger portion of the protein. Given that rchd502 is up-regulated by amechanical force and the rat matrin protein is a nuclear structuralprotein that also binds to DNA, rchd502 may be involved in translating aphysical force on the cell into a program of gene expression.Furthermore, rchd502 is first gene demonstrated to be up-regulated byshear-stress but not by IL-1. It therefore provides an excellent noveltool for diagnosis and treatment of cardiovascular disease.

[0414] The complete sequence of the rchd523 gene reveals that it encodesa novel G protein-coupled receptor protein, consisting of 375 aminoacids and a multiple transmembrane domain sequence motif. The discoveryof such a novel protein is particularly useful in designing treatmentsas well as diagnostic and monitoring systems for cardiovascular disease.In carrying out signal transduction, G proteins play an important earlyrole in the pathways that cause changes in cellular physiology. Therchd523 gene product, therefore, provides an excellent target forintervention in the treatment of cardiovascular disease. Furthermore, asa transmembrane protein, the rchd523 gene product can be readilyaccessed (e.g., by inhibitory compounds during treatment) or detected onthe endothelial cell surface. It, therefore, also provides an excellenttarget for detection of cardiovascular disease states in diagnosticsystems, as well as in the monitoring of the efficacy of compounds inclinical trials. Furthermore, the extracellular domains of the rchd523gene product provide especially efficient screening systems foridentifying compounds that bind to the rchd523 gene product. Suchcompounds can be useful in treating cardiovascular disease by inhibitingrchd523 gene product activity.

[0415] The sequence of the complete coding region of the rchd534 genewas also obtained. The rchd534 gene encodes a novel protein consistingof 235 amino acids.

[0416] Also using the method of Paradigm D, the previously identifiedhuman prostaglandin endoperoxide synthase type II was isolated. Thisgene was previously known to be involved in inflammation, and to beup-regulated by IL-1 (Jones et al., 1993, J. Biol. Chem. 268:9049-9054), but its up-regulation by shear stress was previouslyunknown. This result confirmed the general effectiveness of thetechniques used according to the invention in the detection of genesinvolved cardiovascular disease.

[0417] Furthermore, the up-regulation of these five genes in shearstressed endothelial cells provides a fingerprint for the study ofcardiovascular diseases, including but not limited to atherosclerosis,ischemia/reperfusion, hypertension, and restenosis. The fact that one ofthese genes, rchd502, is not up-regulated under Paradigm C (IL-1induction) provides an extremely useful means of distinguishing andtargeting physiological phenomena specific to shear stress.

[0418] 9.1. Materials and Methods

[0419] Primary cultures of HUVEC's were established from normal termumbilical cords as described (In Progress in Hemostasis and Thrombosis,Vol. 3, P. Spaet, editor, Grune & Stratton Inc., New York, 1-28). Cellswere grown in 20% fetal calf serum complete media (1989, J. Immunol.142: 2257-2263) and passaged 1-3 times before shear stress induction.

[0420] For induction, second passage HUVEC's were plated on tissueculture-treated polystyrene and subjected to 10 dyn/cm2 laminar flow for1 and 6 hr. as described (1994, J. Clin. Invest. 94: 885-891) or 3-10dyn/cm² turbulent flow as previously described (1986 Proc. Natl. Acad.Sci. U.S.A. 83: 2114-2117). RNA was isolated as described, above, inSection 6.1. Differential display, Northern analysis, RT-PCR,subcloning, and DNA sequencing were carried out as described, above, inSection 6.1.2, except that Northern blot hybridizations were carried outas described, above, in Section 8.1.

[0421] For rchd523, the RACE procedure kit was used to obtain the entirecoding region of the rchd523 gene. The procedure was carried outaccording to the manufacturer's instructions (Clonetech, Palo Alto, CA;see also: Chenchik, et al., 1995, CLONTECHniques (X) 1: 5-8; Barnes,1994, Proc. Natl. Acad. Sci. USA 91: 2216-2220; and Cheng et al., Proc.Natl. Acad. Sci. USA 91: 5695-5699). Primers were designed based onamplified rchd523 sequences. Template mRNA was isolated from shearstressed HUVEC's.

[0422] For rchd534, amplified sequences, which contained a portion ofthe gene, were subcloned and then used to retrieve the entire codingregion of the rchd534 gene. Prob's were prepared by isolating thesubcloned insert DNA from vector DNA, and labeling with 32p as describedabove in Section 6.1.2. Labeled insert DNA was used to probe a cDNAlibrary, prepared from mRNA which was isolated from shear stressedHUVEC's as described in this section, above. The cDNA library wasproduced and screened according to well-known methods (Sambrook et al.,1989, supra), using the bacteriophage X-ZAP vector (Stratagene, LaJolla,Calif.). Plaques that were detected by the rchd534 probe were isolatedand sequenced.

[0423] Determination of chromosomal location was carried out accordingto the method described in Section 6.1.3, above. The primers used forrchd523 were (for-atgccgtgtgggttagtc) and (rev-attttatgggaaggtttttaca);and for rchd534 were (for-cttttctgcgtctcccat) and(rev-agacatcagaaactccaacc).

[0424] 9.2. Results

[0425] HUVEC's were subjected to laminar shear stress for 1 or 6 hr. andcompared to static control cells in differential display. As shown inFIG. 19, a band (rchd5o2) is identified which is found in lanes 5,6 (6hr.) but not in lanes 1,2 (control). This band was excised, amplified,and sequenced. Northern analysis using amplified rchd5o2 sequencesrevealed a 4.5 kb band that is up-regulated at 6 hr. compared tocontrols (FIG. 20). When rchd502 probe was hybridized to a Northern blotprepared from IL-1 induced endothelial cells, up-regulation of a 4.5 kbband is not seen (FIG. 21). This result provides the first example of ashear stress-inducible endothelial cell gene that is not regulated byIL-1, indicating that these induction pathways can be separated, and mayprovide for drugs with greater specificity for the treatment ofinflammation and atherosclerosis. Sequencing was done, and the resultingsequence is shown in FIG. 22. When this sequence was compared to thesequence database, an 84% (183/217) sequence similarity with Rat matrinF/G mRNA sequence was obtained.

[0426] Shear stress band rchd505 decreased 1 hr. and 6 hr. after shearstress, as compared to untreated control cells (FIG. 23). Northernanalysis revealed differential expression except that rchd505 wasup-regulated after 1 hr. and 6 hr. shear stress treatment (FIG. 24).This same band was similarly up-regulated in cells treated with IL-1according to Paradigm C (FIG. 25). Sequence analysis revealed thatrchd505 is the previously characterized human endoperoxide synthase typeII. rchd523 was detected under differential display as a bandup-regulated after 1 hr. and 6 hr. shear stress treatment (FIG. 26). The6 hr. up-regulation of rchd523 was confirmed by RT-PCR (FIG. 27).Amplified rchd523 sequences were subcloned, and an isolate was sequencedand designated pRCHD523. The RACE procedure was used to obtain a 2.5 kbcDNA containing the entire coding sequence of the rchd523 gene. The cDNAisolate containing the complete coding sequence of rchd523 is designatedpFCHD523. Sequence analysis revealed that the rchd523 gene productencodes a novel G protein-coupled receptor, consisting of 375 aminoacids and a multiple transmembrane domain sequence motif. A PCRamplification experiment determined that the rchd523 gene is located onhuman chromosome 7. rchd528 was also detected as an up-regulated bandafter 1 hr. and 6 hr. shear stress treatment (FIG. 29). This result wasconfirmed by Northern analysis in which probes of rchd528 amplifiedsequence detected an approximately 5.0 kb message that was up-regulatedmoderately after 1 hr., and up-regulated very strongly after 6 hr. (FIG.30). The amplified sequences were subcloned and sequenced (FIG. 31).Comparison with sequences in the database revealed no homologies betweenrchd528 and any known DNA sequence.

[0427] rchd534 also was detected as being up-regulated in response toshear stress. Northern analysis revealed that rchd534 is stronglyinduced after 6 hours of shear stress treatment (FIG. 34). The amplifiedsequences were subcloned, sequenced, and re-isolated for use as a probefor retrieving full-length rchd534 cDNA. A 3.3 kb X-ZAP clone wassequenced to reveal full-length rchd534 cDNA (FIG. 35). This clonecontaining the entire coding region the rchd534 gene was designatedpFCHD534. The encoded protein consists of 235 amino acids. Comparisonwith sequences in the database revealed no homologies between rchd534and any known DNA sequences. A PCR amplification experiment determinedthat the rchd523 gene is located on human chromosome 15. rchd534 wasalso shown not to be regulated by IL-1 when tested under the conditionsof Paradigm C, as described in Section 8, above. Just like rchd502,rchd534 is an example of a shear stress-inducible endothelial cell genethat is not regulated by IL-1, confirming that these induction pathwayscan be separated, and may provide for drugs with greater specificity forthe treatment of inflammation and atherosclerosis.

10. EXAMPLE: USE OF GENES UNDER PARADIGM A AS SURROGATE MARKERS INCLINICAL TRIALS

[0428] According to the invention, the fingerprint profile derived fromany of the paradigms described in Sections 5.1.1.1 through 5.1.1.6 maybe used to monitor clinical trials of drugs in human patients. Thefingerprint profile, described generally in Section 5.5.4, above,indicates the characteristic pattern of differential gene regulationcorresponding to a particular disease state. Paradigm A, described inSection 5.1.1.1, and illustrated in the example in Section 6, above, forexample, provides the fingerprint profile of monocytes under oxidativestress. This profile gives an indicative reading, therefore, of thephysiological response of monocytes to the uptake of oxidized LDL.Accordingly, the influence of anti-oxidant drugs on the oxidativepotential may be measured by performing differential display on themonocytes of patients undergoing clinical tests.

[0429] 10.1. Treatment of Patients and Cell Isolation

[0430] Test patients may be administered compounds suspected of havinganti-oxidant activity. Control patients may be given a placebo.

[0431] Blood may be drawn from each patient after a 12 hour period offasting and monocytes may be purified as described, above, in Section7.1.1. RNA may be isolated as described in Section 6.1.1, above.

[0432] 10.2. Analysis of Samples

[0433] RNA may be subjected to differential display analysis asdescribed in Section 6.1.2, above. A decrease in the physiologicalresponse state of the monocytes is indicated by a decreased intensity ofthose bands that were up-regulated by oxidized LDL under Paradigm A, andan increased intensity of those bands that were down-regulated byoxidized LDL under Paradigm A, as described in Section 6.2, above.

11. EXAMPLE: IMAGING OF A CARDIOVASCULAR DISEASE CONDITION

[0434] According to the invention, differentially expressed geneproducts which are localized on the surface of affected tissue may beused as markers for imaging the diseased or damaged tissue. Conjugatedantibodies that are specific to the differentially expressed geneproduct may be administered to a patient or a test animal intravenously.This method provides the advantage of allowing the diseased or damagedtissue to be visualized non-invasively.

[0435] 11.1. Monoclonal Conjugated Antibodies

[0436] The differentially expressed surface gene product, such as therchd523 gene product, is expressed in a recombinant host and purifiedusing methods described in Section 5.4.2, above. Preferably, a proteinfragment comprising one or more of the extracellular domains of therchd523 product is produced. Once purified, it is be used to produceF(ab′)₂ or Fab fragments, as described in Section 5.4.3, above. Thesefragments are then labelled with technetium-99m (^(99m)Tc) using aconjugated metal chelator, such as DTPA as described in section 5.8.3,above.

[0437] 11.2. Administration and Detection of Imaging Agents

[0438] Labeled MAb may be administered intravenously to a patient beingdiagnosed for atherosclerosis, restenosis, or ischemia/reperfusion.Sufficient time is allowed for the detectably-labeled antibody tolocalize at the diseased or damaged tissue site (or sites), and bind tothe rchd523 gene product. The signal generated by the label is detectedby a photoscanning device. The detected signal is then converted to animage of the tissue, revealing cells, such as endothelial cells, inwhich rchd523 gene expression is up-regulated.

12. EXAMPLE: SCREENING FOR LIGANDS OF THE rchd 523 GENE PRODUCT ANDANTAGONISTS OF rchd523 GENE PRODUCT-LIGAND INTERACTION

[0439] The rchd523 gene product is a member of the G protein-coupledreceptor protein family, containing multiple transmembrane domains. Thereceptor binding activity of this protein family is detected by assayingfor Ca²⁺ mobility through the membrane of cells in which the receptorgene is expressed. This assay, described below, is used to identifyligands that bind to the rchd523 gene product receptor. Establishingthis ligand-receptor activity then provides for a screen in whichantagonists of the ligand-receptor interaction are identified. Anantagonist is detected by its ability to inhibit the Ca² mobilityinduced by ligand-receptor binding. Such antagonists, therefore, providecompounds that are useful in the treatment of cardiovascular disease, bycounteracting the activity of the product of this target gene which isup-regulated in the disease state.

[0440] Binding of ligand to the rchd523 gene product is measured asfollows. The cDNA containing the entire coding region of the rchd523gene is removed from pFCHD523 and placed under the control of a promoterthat is highly expressed in mammalian cells in an appropriate expressionvector. The resulting construct is transfected into myeloma cells, whichare then loaded with FURA-2 or INDO-1 by standard techniques. Ligandsare added to the cell culture to test their ability to bind to therchd523 receptor in a manner that triggers signal transduction, asmeasured by Ca²⁺ mobilization across the cell membrane. Mobilization ofCa² induced by ligand is measured by fluorescence spectroscopy asdescribed in Grynkiewicz et al., 1985, J. Biol. Chem. 260:3440. Ligandsthat react with the target gene product receptor domain are identifiedby their ability to produce a fluorescent signal. Their receptor bindingactivities are quantified and compared by measuring the level offluorescence produced over background.

[0441] Candidate antagonists are then screened for their ability tointerfere with ligand-receptor binding. Myeloma transfectants expressingrchd523 gene product are treated with ligand alone, and ligand in thepresence of candidate antagonist. Candidate antagonists that cause areduction in the fluorescence signal are designated antagonists of theligand-rchd523 receptor interaction.

13. DEPOSIT OF MICROORGANISMS

[0442] The following microorganisms were deposited with the AgriculturalResearch Service Culture Collection (NRRL), Peoria, Illinois, on January11, 1995 and assigned the indicated-accession numbers: MicroorganismNRRL Accession No. RCHD005 B-21376 RCHD024 B-21377 RCHD032 B-21378RCHD036 B-21379 RCHD502 B-21380 RCHD523 B-21381 RCHD528 B-21382

[0443] The following microorganisms were deposited with the AgriculturalResearch Service Culture Collection (NRRL), Peoria, Ill., on Jun. 6,1995 and assigned the indicated accession numbers: Microorganism NRRLAccession No. FCHD523 FCHD534

[0444] The present invention is not to be limited in scope by thespecific embodiments described herein, which are intended as singleillustrations of individual aspects of the invention, and functionallyequivalent methods and components are within the scope of the invention.Indeed, various modifications of the invention, in addition to thoseshown and described herein will become apparent to those skilled in theart from the foregoing description and accompanying drawings. Suchmodifications are intended to fall within the scope of the appendedclaims.

1 38 288 base pairs nucleic acid both unknown cDNA NO 1 GGCTTAGATGCAGCCTGCAA ATTAAACTTT GATTTTTCAT CTTGTGAAAG CAGTCCTTGT 60 TCCTATGGCCTAATGAACAA CTTCCAGGTA ATGAGTATGG TGTCAGGATT TACACCACTA 120 ATTTCTGCAGGTATATTTTC AGCCACTCTT TCTTCAGCAT TAGCATCCCT AGTGAGTGCT 180 CCCAAAATATTTCAGGCTCT ATGTAAGGAC AACATCTACC CAGCTTTCCA GATGTTTGCT 240 AAAGGTTATGGGAAAAATAA TGAACCTCTT CGTGGCTGCA TCTAAGCC 288 178 base pairs nucleicacid both unknown cDNA NO 2 AAAAATAAAT AAATTAAAGT CTGAGACCAA TTTGCCACTGTGAATATAAG CACATTAACC 60 CCAGGAGGAG CCAAGAACTA CACAAACCTC TCTATGAGAATTTACCAGTC TTCTTTCATT 120 TGGCAAGAAA AAGCTCAGGA AAATTTGCTT GTTTAAATTCTATGAGCCTA GTCTATGG 178 101 base pairs nucleic acid both unknown cDNA NO3 GGGTAATTCA TTAATTACAC TTTAAAATTG GAAAGTGGGA TAAGAAATCT AAAGTAAACC 60AGCTTATCTT TGAAACAATA TTATTTTGAA ATTGGCTTTA A 101 184 base pairs nucleicacid both unknown cDNA NO 4 GGCTTGGTGG TGATGCCTAC AAGAAATGTT TACATACAAACACTCTATAC ATCTAACTCC 60 CGAAAAAGGA CCAGCTATTT CGGCAACAGA AAAAAGACAAGCATTTCAGA GGAGCGTTGC 120 TTTCCTTAAA GACCTAACTC ACTTAAGTCT TACAAACAGAAATAACAAGG AGGACAATTT 180 TCTA 184 284 base pairs nucleic acid bothunknown cDNA NO 5 CTTGGGGATG CTGTTTGGAG GAATCCTCAT GAAGCGCTTT GTTTTCTCTCTACAAGCCAT 60 TCCCCGCATA GCTACCACCA TCATCACCAT CTCCATGATC CTTTGTGTTCCTTTGTTCTT 120 CATGGGATGC TCCACCCCAA CTGTGGCCGA AGTCTACCCC CCTAGCACATCAAGTTCTAT 180 ACATCCGCAG TCTCCTGCCT GCCGCAGGGA CTGCTCGTGC CCAGATTCTATCTTCCACCC 240 GGTCTGTGGA GACAATGGAA TCGAGTACCT CTCCCCTTGC CATG 284 2582base pairs nucleic acid both unknown cDNA NO 6 GGCTTACCAT CGATGCGGCCGCGGATCCAG GGCTCAGAGG GAGGACGCAC CCGCCAGCCA 60 GCCGGGAACC TTCCCTCGCGGGCTCCCAGG GCGGGTCTCT TCCTCTCTCT AGCCCTGCTC 120 AGGCATTCGG CAGGTCCAGCAGAGGTACAC CTCCTGCAGC GGGTTCCAAG TGCACCTCCA 180 GCCTGATGGA CCTGACCAAGGAGGCTTCCA GGAGCACAGA AGGGGCTGCA ACCCAGGTAC 240 CCAGAGAGTG AGCAGCTCCACGCGGGACTG TGCACGGTGG CCGACACCCG CAGGGACGCC 300 CACCGGACGA GCACGCGGAGGGCCCTCGCC TCCACGGATG CACCATGCCG GTGTGAGGAG 360 CATCTGTTCT TCCCACTCTCTGCAGTTAAC AAACCCAACC CAAACCACCA CAGGTGCTCC 420 TCCTGGGGAG TTTCCTGTCTGACAAATGCC AGGCTCACTT CAAGGAGAAT CACGCTTCTT 480 TCTAAAGATG GATTCACCATTTAAAACAGA GCTCTGGGAG CCTTTCGGCA AATCTTGAAA 540 GCTGCACGGC GCAGAGACATGGATGTGACT TCCCAAGCCC GGGGCGTAGG CCTGGAGATG 600 TACCCAGGCA CCGCGCAGCCTGCGGCCCCC AACACCACCT CCCCCGAGCT CAACCTGTCC 660 CACCCGCTCC TGGGCACCGCCCTGGCCAAT GGGACAGGTG AGCTCTCGGA GCACCAGCAA 720 TACGTGATCG GCCTGTTCCTCTCGTGCCTC TACACCATCT TCCTCTTCCC CATCGGCTTT 780 GTGGGCAACA TCCTGATCCTGGTGGTGAAC ATCAGCTTCC GCGAGAAGAT GACCATCCCC 840 GACCTGTACT TCATCAACCTGGCGGTGGCG GACCTCATCC TGGTGGCCGA CTCCCTCATT 900 GAGGTGTTCA ACCTGCACGAGCGGTACTAC GACATCGCCG TCCTGTGCAC CTTCATGTCG 960 CTCTTCCTGC GGGTCAACATGTACAGCAGC GTCTTCTTCC TCACCTGGAT GAGCTTCGAC 1020 CGCTACATCG CCCTGGCCAGGGCCATGCGC TGCAGCCTGT TCCGCACCAA GCACCACGCC 1080 CGGCTGAGCT GTGGCCTCATCTGGATGGCA TCCGTGTCAG CCACGCTGGT GCCCTTCACC 1140 GCCGTGCACC TGCAGCACACCGACGAGGCC TGCTTCTGTT TCGCGGATGT CCGGGAGGTG 1200 CAGTGGCTCG AGGTCACGCTGGGCTTCATC GTGCCCTTCG CCATCATCGG CCTGTGCTAC 1260 TCCCTCATTG TCCGGGTGCTGGTCAGGGCG CACCGGCACC GTGGGCTGCG GCCCCGGCGG 1320 CAGAAGGCGC TCCGCATGATCCTCGCAGTG GTGCTGGTCT TCTTCGTCTG CTGGCTGCCG 1380 GAGAACGTCT TCATCAGCGTGCACCTCCTG CAGCGGACGC AGCCTGGGGC CGCTCCTTGC 1440 AAGCAGTCTT TCCGCCATGCCCACCCCCTC ACGGGCCACA TTGTCAACCT CGCCGCCTTC 1500 TCCAACAGCT GCCTAAACCCCCTCATCTAC AGCTTTCTCG GGGAGACCTT CAGGGACAAG 1560 CTGAGGCTGT ACATTGAGCAGAAAACAAAT TTGCCGGCCC TGGACCGCTT CTGTCACGCT 1620 GCCCTGAAGG CCGTCATTCCAGACAGCACC GAGCAGTCGG ATGTGAGGTT CAGCAGTGCC 1680 GTGTAGACAG CCTTGGCCGCATAGGCCCAG CCAGGGTGTG ACTCGGGAGC TGCACACACC 1740 TGGGTGGACA CAAGGCACGGCCACGTCATG TCTCTAAACT GCGGTCAGAT GTGGCTTCTG 1800 GCTCCTCGGG CCTCGCGAGGGTCACGCTTG CCTGGTCACC CTGGGGCTGC TTAGGAAACC 1860 TCAGGACTGG TCACCTTGCACTCCTCACAC AGAATTGCTA CAATCCCAAA GCGCTCGCCC 1920 CGCAGGGTCC AAAGGCCAGCGGTGACCAGC CTGTCACCCA GCTCCTCCCC GCCAACCCTG 1980 CCTGCCGCTG CACCTGCCCGCTGCTGCAGG AAACATTTCT GACACCGTCG ACCAGGAAAG 2040 CCACACGGAG AGGCCACTGTGGGTGAAGCG CCTCAGTTAC ACAGGAACCC TAAAGCAAAT 2100 CTGCCACCGT GGGGGAACTGACGCTGGAGA TGCAAGGTGC TGGTGGGTCT GAGCTGGACG 2160 TCGCGGTGTG TCCTCTGTGCCCACGGTCTG AGCTAGCTAG CGCACCGCCG AGTTAAAGAG 2220 GAGAAGGAAA ACATGCTGCTCTGGTGCACG CCTGAGCGTC CTCCATCTTC CAGGATGGCA 2280 GCAATGGCGC TGTGCGGCCTCACCAGGCCC ACGAGGAGCA GCAGCGCTCG GCCCGGAGCA 2340 GCAGGAAGGC CCCTCTGTGGAGCGCCCGCC GTCTGCTCCG GGGTGGTTCA GTCACTGCTT 2400 GTTGACATCA ACATGGCAATTGCACTCATG TGGACTGGGA CCGTGCGAGC TGCCGTGTGG 2460 GTTAGTCGGG TGCCAGGACAATGAAATACT CCAGCACCTG TGGCTGACGA ATTCGTTTCT 2520 ACAGAAGTAA CAGCTGGGGACAACTGCGAT GATGATGTAA AAACCTTCCC ATAAAATAAG 2580 CC 2582 128 base pairsnucleic acid both unknown cDNA NO 7 GGGAGGTGGG CTCCTGCTCA TCCTAGGCATCGCACTGATT GTTACCTGTT GCAGAAAGAA 60 TAAAAATGAC ATAAGCAAAC TCATCTTCAAAAGTGGAGAT TTCCAAATGT CCCCGTATGC 120 TGAATACC 128 13 base pairs nucleicacid single linear DNA (genomic) NO misc_feature 12 8 TTTTTTTTTT TNG 1310 base pairs nucleic acid single linear DNA (genomic) NO 9 AGCATGGCTC10 23 base pairs nucleic acid single linear DNA (genomic) NO 10CACCCCTGGC ATCTTCTCCT TCC 23 24 base pairs nucleic acid single linearDNA (genomic) NO 11 ATCCTCCCCC AGTTCACCCC ATCC 24 21 base pairs nucleicacid single linear DNA (genomic) NO 12 CCTGATAGAT GGGCACTGTG T 21 22base pairs nucleic acid single linear DNA (genomic) NO 13 GAACACGGCATTGTCACTAA CT 22 22 base pairs nucleic acid single linear DNA (genomic)NO 14 AAGTCGCGCC CGCCCCTGAA AT 22 24 base pairs nucleic acid singlelinear DNA (genomic) NO 15 GATCCCTGGC CACCGTCCGT CTGA 24 17 base pairsnucleic acid single linear DNA (genomic) NO 16 ACCCTGAAGT ACCCCAT 17 17base pairs nucleic acid single linear DNA (genomic) NO 17 TAGAAGCATTTGCGGTG 17 10 base pairs nucleic acid single linear DNA (genomic) NO 18AGATGCAGCC 10 13 base pairs nucleic acid single linear DNA (genomic) NOmisc_feature 12 19 TTTTTTTTTT TNA 13 10 base pairs nucleic acid singlelinear DNA (genomic) NO 20 TCTCCCTCAG 10 13 base pairs nucleic acidsingle linear DNA (genomic) NO misc_feature 12 21 TTTTTTTTTT TNC 13 10base pairs nucleic acid single linear DNA (genomic) NO 22 TGGAGAGCAG 1023 base pairs nucleic acid single linear DNA (genomic) NO 23 ATTTATAAAGGGGTAATTCA TTA 23 22 base pairs nucleic acid single linear DNA (genomic)NO 24 TTAAAGCCAA TTTCAAAATA AT 22 10 base pairs nucleic acid singlelinear DNA (genomic) NO 25 GGTGGTGATG 10 10 base pairs nucleic acidsingle linear DNA (genomic) NO 26 GGTGCGGGAA 10 10 base pairs nucleicacid single linear DNA (genomic) NO 27 ACATGCCGTG 10 18 base pairsnucleic acid single linear DNA (genomic) NO 28 ATGCCGTGTG GGTTAGTC 18 22base pairs nucleic acid single linear DNA (genomic) NO 29 ATTTTATGGGAAGGTTTTTA CA 22 10 base pairs nucleic acid single linear DNA (genomic)NO 30 AATGCGGGAG 10 13 base pairs nucleic acid single linear DNA(genomic) NO misc_feature 12..13 31 TTTTTTTTTT TNN 13 13 base pairsnucleic acid single linear DNA (genomic) NO 32 TTTTTTTTTT TCC 13 10 basepairs nucleic acid single linear DNA (genomic) NO 33 GTGACATGCC 10 20base pairs nucleic acid single linear DNA (genomic) NO 34 CATGCCTGTAGAAAAAGGTT 20 20 base pairs nucleic acid single linear DNA (genomic) NO35 CTTCATAGAA TCTAAGCCTA 20 3083 base pairs nucleic acid both unknowncDNA misc_feature 16 misc_feature 30 misc_feature 2911 36 GAATTCGGCACGAGGMCAGG AGCTCCTTTW CTGCGTCTCC CATCATGGGG CTTAGGGTTG 60 AGTCTTCAGGTTCTGGGGGC AGGAAGGACG GGCACTCAGG AGGCCCCCTC CCCATCCACA 120 GCCCCTCTTTGGGAGGGGGG AAACTTGGCA ACCCGGGAGG CATGTGGATC TTTTCCTAAG 180 CAAGATGCTGAGCTGGAAAG ATGGGGGTGT AAGGTAATGT CCCAAACTGA AACTTTGCCA 240 GGCACTGGGAGAGGCTGTGA ACTCTTTTCT GGCTTTAGAA TTTAGGTCTA GATCCCAAAA 300 GGCTAAGTACCCCCTGGGGG CTAACCAGAG GCATGCCTGG GCTGAGCTGA ACCTTCTGGT 360 GCACTGGCCCCTGGCTGACT GCTCTTCTGC AGGAAGTTGG AGGAGATTCC TGAAGTTGAT 420 TCCTCAGGCTGGATGTCCAA GGGGGTTGGA GTTTCTGATG TCTTTCTGTC TCCCTCTCTT 480 TTCTTTCTCTCCCTACCAGG TCCACTTCTT TCAGAGGGGC CTGCGGTGCT CTAAAAGTTC 540 TCCTGTTAAAGTTTAGAGCA AATTGGTTAT TATTTTAAAA TCAATAAAAC TTTTAAAAGT 600 ACTAAGACAACTTCTAAGAG GGGAGTGGAC AGAGGGCCTG GTGGCAGCTC ACAGTTTCTT 660 TTCTGACCTTTGGTCTCACC CACCAAGTGT CCCACCTGAG TGCCCACCTT GCCCACCTGA 720 GGTAATGCCCTGGGGCTCCA CCAGTCCAGA TCCACAGGGC GCAGCCATGT GGGAGTGGCG 780 GCTGATTGTTACCCAGTAGT GTTGATAGCA CATTATTCAT AACAGCCAAA GAGAGGAAGC 840 AACCCAAATGTCCATTAGCT GATAAATGGA TAAATGAAAT ATGGTACGTC CGAAGAATGG 900 AATATCATTCACCCATGAAA AAGAACGAAG TCCAGCACCA AAACGTGCTA CAACATGGAT 960 GAACTTCGATGACTTTGTGC CACATGAAAG AAGAAGCCAG CCACAAAAGG CCATATATTG 1020 TATGAAATGAAATGTCCAGA ATGGGCAAAC CCATAGAGAC ACAAAAATCT CCGCCACCTC 1080 CCTACTCTCGGCTGTCTCCT CGCGACGAGT ACAAGCCACT GGATCTGTCC GATTCCACAT 1140 TGTCTTACACTGAAACGGAG GCTACCAACT CCCTCATCAC TGCTCCGGGT GAATTCTCAG 1200 ACGCCAGCATGTCTCCGGAC GCCACCAAGC CGAGCCACTG GTGCAGCGTG GCGTACTGGG 1260 AGCACCGGACGCGCGTGGGC CGCCTCTATG CGGTGTACGA CCAGGCCGTC AGCATCTTCT 1320 ACGACCTACCTCAGGGCAGC GGCTTCTGCC TGGGCCAGCT CAACCTGGAG CAGCGCAGCG 1380 AGTCGGTGCGGCGAACGCGC AGCAAGATCG GCTTCGGCAT CCTGCTCAGC AAGGAGCCCG 1440 ACGGCGTGTGGGCCTACAAC CGCGGCGAGC ACCCCATCTT CGTCAACTCC CCGACGCTGG 1500 ACGCGCCCGGCGGCCGCGCC CTGGTCGTGC GCAAGGTGCC CCCCGGCTAC TCCATCAAGG 1560 TGTTCGACTTCGAGCGCTCG GGCCTGCAGC ACGCGCCCGA GCCCGACGCC GCCGACGGCC 1620 CCTACGACCCCAACAGCGTC CGCATCAGCT TCGCCAAGGG CTGGGGGCCC TGCTACTCCC 1680 GGCAGTTCATCACCTCCTGC CCCTGCTGGC TGGAGATCCT CCTCAACAAC CCCAGATAGT 1740 GGCGGCCCCGGCGGGAGGGG CGGGTGGGAG GCCGCGGCCA CCGCCACCTG CCGGCCTCGA 1800 GAGGGGCCGATGCCCAGAGA CACAGCCCCC ACGGACAAAA CCCCCCAGAT ATCATCTACC 1860 TAGATTTAATATAAAGTTTT ATATATTATA TGGAAATATA TATTATACTT GTAATTATGG 1920 AGTCATTTTTACAATGTAAT TATTTATGTA TGGTGCAATG TGTGTATATG GACAAAACAA 1980 GAAAGACGCACTTTGGCTTA TAATTCTTTC AATACAGATA TATTTTCTTT CTCTTCCTCC 2040 TTCCTCTTCCTTACTTTTTA TATATATATA TAAAGAAAAT GATACAGCAG AGCTAGGTGG 2100 AAAAGCCTGGGTTTGGTGTA TGGTTTTTGA GATATTAATG CCCAGACAAA AAGCTAATAC 2160 CAGTCACTCGATAATAAAGT ATTCGCATTA TAGTTTTTTT TAAACTGTCT TCTTTTTACA 2220 AAGAGGGGCAGGTAGGGCTT CAGCGGATTT CTGACCCATC ATGTACCTTG AAACTTGACC 2280 TCAGTTTTCAAGTTTTACTT TTATTGGATA AAGACAGAAC AAATTGAAAA GGGAGGAAAG 2340 TCACATTTACTCTTAAGTAA ACCAGAGAAA GTTCTGTTGT TCCTTCCTGC CCATGGCTAT 2400 GGGGTGTCCAGTGGATAGGG ATGGCGGTGG GGAAAAGGAG AATACACTGG CCATTTATCC 2460 TGGACAAGCTCTTCCAGTCT GATGGAGGAG GTTCATGCCC TAGCCTAGAA AGGCCCAGGT 2520 CCATGACCCCCATCTTTGAG TTATGAGCAA GCTAAAAGAA GACACTATTT CTCACCATTT 2580 TGTGGAAATGGCCTGGGGAA CAAAGACTGA AATGGGCCTT GAGCCCACCT GCTACCTTGC 2640 AGAGAACCATCTCGAGCCCC GTAGATCTTT TTAGGACCTC CACAGGCTAT TTCCCACCCC 2700 CCAGCCAAAAATAGCTCAGA ATCTGCCCAT CCAGGGCTGT ATTAATGATT TATGTAAAGG 2760 CAGATGGTTTATTTCTACTT TGTAAAAGGG AAAAGTTGAG GTTCTGGAAG GATAAATGAT 2820 TTGCTCATGAGACAAAATCA AGGTTAGAAG TTACATGGAA TTGTAGGACC AGAGCCATAT 2880 CATTAGATCAGCTTTCTGAA GAATATTCTC MAAAAAAGAA AGTCTCCTTG GCCAGATAAC 2940 TAAGAGGAATGTTTCATTGT ATATCTTTTT TCTTGGAGAT TTATATTAAC ATATTAAGTG 3000 CTCTGAGAAGTCCTGTGTAT TATCTCTTGC TGCATAATAA ATTATCCCCA AACTTAAAAA 3060 AAAAAAAAAAAAAAAAACTC GAG 3083 235 amino acids amino acid <Unknown> unknown protein37 Met Ser Arg Met Gly Lys Pro Ile Glu Thr Gln Lys Ser Pro Pro Pro 1 510 15 Pro Tyr Ser Arg Leu Ser Pro Arg Asp Glu Tyr Lys Pro Leu Asp Leu 2025 30 Ser Asp Ser Thr Leu Ser Tyr Thr Glu Thr Glu Ala Thr Asn Ser Leu 3540 45 Ile Thr Ala Pro Gly Glu Phe Ser Asp Ala Ser Met Ser Pro Asp Ala 5055 60 Thr Lys Pro Ser His Trp Cys Ser Val Ala Tyr Trp Glu His Arg Thr 6570 75 80 Arg Val Gly Arg Leu Tyr Ala Val Tyr Asp Gln Ala Val Ser Ile Phe85 90 95 Tyr Asp Leu Pro Gln Gly Ser Gly Phe Cys Leu Gly Gln Leu Asn Leu100 105 110 Glu Gln Arg Ser Glu Ser Val Arg Arg Thr Arg Ser Lys Ile GlyPhe 115 120 125 Gly Ile Leu Leu Ser Lys Glu Pro Asp Gly Val Trp Ala TyrAsn Arg 130 135 140 Gly Glu His Pro Ile Phe Val Asn Ser Pro Thr Leu AspAla Pro Gly 145 150 155 160 Gly Arg Ala Leu Val Val Arg Lys Val Pro ProGly Tyr Ser Ile Lys 165 170 175 Val Phe Asp Phe Glu Arg Ser Gly Leu GlnHis Ala Pro Glu Pro Asp 180 185 190 Ala Ala Asp Gly Pro Tyr Asp Pro AsnSer Val Arg Ile Ser Phe Ala 195 200 205 Lys Gly Trp Gly Pro Cys Tyr SerArg Gln Phe Ile Thr Ser Cys Pro 210 215 220 Cys Trp Leu Glu Ile Leu LeuAsn Asn Pro Arg 225 230 235 375 amino acids amino acid <Unknown> unknownprotein 38 Met Asp Val Thr Ser Gln Ala Arg Gly Val Gly Leu Glu Met TyrPro 1 5 10 15 Gly Thr Ala Gln Pro Ala Ala Pro Asn Thr Thr Ser Pro GluLeu Asn 20 25 30 Leu Ser His Pro Leu Leu Gly Thr Ala Leu Ala Asn Gly ThrGly Glu 35 40 45 Leu Ser Glu His Gln Gln Tyr Val Ile Gly Leu Phe Leu SerCys Leu 50 55 60 Tyr Thr Ile Phe Leu Phe Pro Ile Gly Phe Val Gly Asn IleLeu Ile 65 70 75 80 Leu Val Val Asn Ile Ser Phe Arg Glu Lys Met Thr IlePro Asp Leu 85 90 95 Tyr Phe Ile Asn Leu Ala Val Ala Asp Leu Ile Leu ValAla Asp Ser 100 105 110 Leu Ile Glu Val Phe Asn Leu His Glu Arg Tyr TyrAsp Ile Ala Val 115 120 125 Leu Cys Thr Phe Met Ser Leu Phe Leu Arg ValAsn Met Tyr Ser Ser 130 135 140 Val Phe Phe Leu Thr Trp Met Ser Phe AspArg Tyr Ile Ala Leu Ala 145 150 155 160 Arg Ala Met Arg Cys Ser Leu PheArg Thr Lys His His Ala Arg Leu 165 170 175 Ser Cys Gly Leu Ile Trp MetAla Ser Val Ser Ala Thr Leu Val Pro 180 185 190 Phe Thr Ala Val His LeuGln His Thr Asp Glu Ala Cys Phe Cys Phe 195 200 205 Ala Asp Val Arg GluVal Gln Trp Leu Glu Val Thr Leu Gly Phe Ile 210 215 220 Val Pro Phe AlaIle Ile Gly Leu Cys Tyr Ser Leu Ile Val Arg Val 225 230 235 240 Leu ValArg Ala His Arg His Arg Gly Leu Arg Pro Arg Arg Gln Lys 245 250 255 AlaLeu Arg Met Ile Leu Ala Val Val Leu Val Phe Phe Val Cys Trp 260 265 270Leu Pro Glu Asn Val Phe Ile Ser Val His Leu Leu Gln Arg Thr Gln 275 280285 Pro Gly Ala Ala Pro Cys Lys Gln Ser Phe Arg His Ala His Pro Leu 290295 300 Thr Gly His Ile Val Asn Leu Ala Ala Phe Ser Asn Ser Cys Leu Asn305 310 315 320 Pro Leu Ile Tyr Ser Phe Leu Gly Glu Thr Phe Arg Asp LysLeu Arg 325 330 335 Leu Tyr Ile Glu Gln Lys Thr Asn Leu Pro Ala Leu AspArg Phe Cys 340 345 350 His Ala Ala Leu Lys Ala Val Ile Pro Asp Ser ThrGlu Gln Ser Asp 355 360 365 Val Arg Phe Ser Ser Ala Val 370 375

What is claimed is:
 1. An isolated nucleic acid containing the following nucleotide sequence: rchd005 (SEQ ID NO.:1), rchd024 (SEQ ID NO.:2), rchd032 (SEQ ID NO.:3), rchd036 (SEQ ID NO.:4), rchd502 (SEQ ID NO.:5), rchd523 (SEQ ID NO.:6), rchd528 (SEQ ID NO.:7), or rchd534 (SEQ ID NO.:36). or the nucleotide sequence of a gene or gene fragment contained in the following clone as deposited with the NRRL: pRCHD005 (in NRRL Accession No. B-21376), pRCHD024 (in NRRL Accession No. B-21377), pRCHD032 (in NRRL Accession No. B-21378), pRCHD036 (in NRRL Accession No. B-21379), PRCHD502 (in NRRL Accession No. B-21380), PRCHD523 (in NRRL Accession No. B-21381), pFCHD523 (in NRRL Accession No. ), PRCHD528 (in NRRL Accession No. B-21382), or pFCHD534 (in NRRL Accession No. ).
 2. An isolated nucleic acid which hybridizes under stringent conditions to the nucleotide sequence of claim 1 or its complement, or to the gene or gene fragment contained in the clone of claim 1 as deposited with the NRRL.
 3. An isolated nucleic acid which encodes an amino acid sequence encoded by the nucleotide sequence of claim 1 or its complement, or the gene or gene fragment contained in the clone of claim 1 as deposited with the NRRL.
 4. A nucleotide vector containing the nucleotide sequence of claim 1, 2 or
 3. 5. An expression vector containing the nucleotide sequence of claim 1, 2 or 3 in operative association with a nucleotide regulatory element that controls expression of the nucleotide sequence in a host cell.
 6. A genetically engineered host cell containing the nucleotide sequence of claim 1, 2 or
 3. 7. A genetically engineered host cell containing the nucleotide sequence of claim 1, 2 or 3 in operative association with a nucleotide regulatory element that controls expression of the nucleotide sequence in the host cell.
 8. A substantially pure gene product encoded by the nucleic acid of claim 1, 2, or
 3. 9. An antibody that immunospecifically binds the gene product of claim
 8. 10. A transgenic animal in which the nucleic acid of claim 1, 2 or 3 is an expressed transgene contained in the genome of the animal.
 11. A transgenic animal in which expression of genomic sequences encoding the gene product of claim 8 is prevented or suppressed.
 12. A method for diagnosing cardiovascular disease, comprising detecting, in a patient sample, a gene or its gene product which is differentially expressed in cardiovascular disease states.
 13. The method of claim 12 in which the cardiovascular disease is atherosclerosis.
 14. The method of claim 12 in which the cardiovascular disease is ischemia/reperfusion.
 15. The method of claim 12 in which the cardiovascular disease is hypertension.
 16. The method of claim 12 in which the cardiovascular disease is restenosis.
 17. The method of claim 12 in which the gene is up-regulated in individuals genetically predisposed to cardiovascular disease.
 18. The method of claim 17 in which the gene encodes a Na—K—Cl cotransporter protein homologue, an rchd024 protein, and rchd032 protein, an rchd036 protein, a homolog of rat matrin F/G protein, an endoperoxide synthase type II protein, an rchd523 protein, an rchd528 protein, or an rchd534 protein.
 19. The method of claim 12 in which the gene is down-regulated in individuals genetically predisposed to cardiovascular disease.
 20. The method of claim 19 in which the gene encodes a glutathione peroxidase protein or a Bcl-2 protein.
 21. The method of claim 12 in which the gene is up-regulated by treatment with IL-1.
 22. The method of claim 21 in which the gene encodes an Na—K—Cl cotransporter protein homologue, an rchd024 protein, an rchd032 protein, an rchd036 protein, or an endoperoxide synthase type II protein.
 23. The method of claim 12 in which the gene is up-regulated by treatment with shear stress.
 24. The method of claim 23 in which the gene encodes an Na—K—Cl cotransporter protein homologue, an rchd024 protein, a rat matrin F/G protein homologue, an endoperoxide synthase type II protein, an rchd523 protein, an rchd528 protein, or an rchd534 protein.
 25. The method of claim 12 wherein the gene is down-regulated by treatment of individuals with a high fat/high cholesterol diet.
 26. The method of claim 25 in which the gene encodes a glutathione peroxidase protein or a Bcl-2 protein.
 27. A method for treating cardiovascular disease, comprising administering a compound that modulates the synthesis or expression of a target gene, or the activity of a target gene product to a patient in need of such treatment.
 28. The method of claim 27 in which the cardiovascular disease is atherosclerosis.
 29. The method of claim 27 in which the cardiovascular disease is ischemia/reperfusion.
 30. The method of claim 27 in which the cardiovascular disease is hypertension.
 31. The method of claim 27 in which the cardiovascular disease is restenosis.
 32. The method of claim 27 in which the compound inhibits the expression of the target gene, or the synthesis or activity of the target gene product.
 33. The method of claim 32 in which the gene encodes a Na—K—Cl cotransporter protein homologue, an rchd024 protein, and rchd032 protein, an rchd036 protein, a homolog of rat matrin F/G protein, an endoperoxide synthase type II protein, an chd523 protein, an rchd528 protein, or an rchd534 protein.
 34. The method of claim 27 in which the compound is an ntisense or ribozyme molecule that blocks translation of the arget gene.
 35. The method of claim 34 in which the gene encodes a Na—K—Cl cotransporter protein homologue, an rchd024 protein, and rchd032 protein, an rchd036 protein, a homologue of rat atrin F/G protein, an endoperoxide synthase type II protein, and rchd523 protein, an rchd528 protein, or an rchd534 protein.
 36. The method of claim 27 in which the compound is complementary to the 5′ region of the target gene and blocks transcription via triple helix formation.
 37. The method of claim 36 in which the gene encodes a Na—K—Cl cotransporter protein homologue, an rchd024 protein, and rchd032 protein, an rchd036 protein, a homologue of rat matrin F/G protein, an endoperoxide synthase type II protein, and rchd523 protein, an rchd528 protein, or an rchd534 protein.
 38. The method of claim 27 in which the compound is an antibody that neutralizes the activity of the target gene product.
 39. The method of claim 38 in which the gene product is a Na—K—Cl cotransporter protein homologue, an rchd024 protein, and rchd032 protein, an rchd036 protein, a homologue of rat matrin F/G protein, an endoperoxide synthase type II protein, and rchd523 protein, an rchd528 protein, or an rchd534 protein.
 40. The method of claim 27 in which the compound enhances the expression of the target gene, or the synthesis or activity the target gene product.
 41. The method of claim 40 in which the target gene encodes Bcl-2 or glutathione peroxidase.
 42. A method for treating cardiovascular disease, comprising administering nucleic acid encoding an active target gene product to a patient in need of such treatment.
 43. The method of claim 42 in which the nucleic acid encodes Bcl-2 or glutathione peroxidase.
 44. A method for treating cardiovascular disease, comprising administering an effective amount of a target gene product to a patient in need of such therapy.
 45. The method of claim 44 in which the gene product is Bcl-2 or glutathione peroxidase.
 46. A method of monitoring the efficacy of a compound in clinical trials for the treatment of cardiovascular disease, comprising detecting, in a patient sample, a gene or its gene product which is differentially expressed in cardiovascular disease states.
 47. The method of claim 46 in which the cardiovascular disease is atherosclerosis.
 48. The method of claim 46 in which the cardiovascular disease is ischemia/reperfusion.
 49. The method of claim 46 in which the cardiovascular disease is hypertension.
 50. The method of claim 46 in which the cardiovascular disease is restenosis.
 51. The method of claim 46 in which the gene is up-regulated in individuals genetically predisposed to cardiovascular disease.
 52. The method of claim 51 in which the gene encodes a Na—K—Cl cotransporter protein homologue, an rchd024 protein, and rchd032 protein, an rchd036 protein, a homolog of rat matrin F/G protein, an endoperoxide synthase type II protein, and rchd523 protein, an rchd528 protein, or an rchd534 protein.
 53. The method of claim 46 in which the gene is down-regulated in individuals genetically predisposed to cardiovascular disease.
 54. The method of claim 53 in which the gene encodes a glutathione peroxidase protein or a Bcl-2 protein.
 55. The method of claim 46 in which the gene is up-regulated by treatment with IL-1.
 56. The method of claim 55 in which the gene encodes an Na—K—Cl cotransporter protein homologue, an rchd024 protein, an rchd032 protein, an rchd036 protein, or an endoperoxide synthase type II protein.
 57. The method of claim 46 in which the gene is up-regulated by treatment with shear stress.
 58. The method of claim 57 in which the gene encodes an Na—K—Cl cotransporter protein homologue, an rchd024 protein, a rat matrin F/G protein homologue, an endoperoxide synthase type II protein, an rchd523 protein, an rchd528 protein, or an rchd534 protein.
 59. The method of claim 46 wherein the gene is down-regulated by treatment of individuals with a high fat/high cholesterol diet.
 60. The method of claim 59 in which the gene encodes a glutathione peroxidase protein or a Bcl-2 protein.
 61. A method for identifying a compound that modulates the activity of a multiple transmembrane domain receptor target gene product, comprising: contacting a first cell expressing the multiple transmembrane domain receptor target gene product wtith a test compound and an activator of the multiple transmembrane domain receptor target gene product, measuring the level of intracellular calcium release within the first cell and comparing the level to that of a second multiple transmembrane domain receptor target gene product-expressing cell which has been contacted with the activator but not with the test compound so that if the level of intracellular calcium release within the first cells differs from that of the second cell, a compound which modulates the activity of a multiple transmembrane domain receptor target gene product has been identified.
 62. The method of claim 61 wherein the multiple transmembrane domain receptor target gene product is an rchd523 gene product.
 63. The method of claim 61 wherein the cell is a Xenopus oocyte cell.
 64. The method of claim 61 wherein the cell is a myeloma cell.
 65. The method of claim 18 in which the gene encodes an rchd523 protein.
 66. The method of claim 18 in which the gene encodes an rchd534 protein. 